KR101439719B1 - Antibodies against csf-1r - Google Patents

Abstract

The present invention provides a human antibody that binds to human CSF-1R with high affinity. The antibodies of the present invention have significant advantages over antibodies known in the art by their multifunctional properties: inhibition of CSF-1R signaling in cells including tumors, macrophages and monocytes, internalization of CSF-1R degradation And stimulation of induction and ADCC. They can also be combined alone or in combination with docetaxel, paclitaxel, doxorubicin or Herceptin ^® it has been found to be effective in the treatment of leukemia, breast cancer, endometrial cancer and prostate cancer.

Description

Antibodies against CSF-1R {ANTIBODIES AGAINST CSF-1R}

This application claims priority to U.S. Provisional Application No. 61 / 319,896, filed April 1, 2010.

The present invention relates to the field of immunology and cancer therapy. More specifically, the invention relates to human antibodies that bind to the human colony stimulating factor-1 receptor (CSF-IR).

(human CSF-1R variant; SEQ ID NO: 15) (human CSF-1R; SEQ ID NO: 16) which is encoded by the c-fms gene (also known as M-CSFR or CD-115) ; Uniprot registration # P07333) is a tyrosine kinase receptor that is selectively expressed on the macrophage granulocyte lineage of normal individuals and on tumor cells of cancer. (Human CSF-1; SEQ ID NO: 17) (Uniproth registration # P09603) (also known as M-CSF and IL) binding to the extracellular domain of CSF-1R, colony stimulating factor-1 -34) (human IL-34; SEQ ID NO: 18) (Uniproth registration # Q6ZMJ4). Upon CSF-1 or IL-34 binding, CSF-1R is dimerized leading to phosphorylation and activation of receptor trans-phosphorylation and downstream signaling molecules, such as MAPK and Akt. The phosphorylation of CSF-1R leads to the following: (1) proliferation and differentiation of macrophages from hematopoietic stem cells and (2) survival of macrophages and their migration into various organs and tissues, particularly tumor metastases, in the body. CSF-IR can also be expressed on the surface of tumor cells.

Antibodies to murine CSF-1R are not cross-reactive in humans and therefore would be ineffective therapeutics for treating cancer in humans.

Human antibodies to CSF-1R are disclosed in PCT publication WO2009 / 026303 (Brasel, et al.). Such antibodies do not induce antibody-dependent cell-mediated cytotoxicity (ADCC) activity on cells bound by the antibody. When an antibody binds to an antigen-expressing cell (target cell), ADCC occurs to produce the Fc region of the antibody available for binding to the Fc receptor on natural killer cells, monocytes, neutrophils and dendritic cells (effector cells). Antibodies disclosed in Brasel can not bring together target cells and effector cells to initiate the death of target cells.

Antibodies to ligands are not cross-reactive. Thus, antibodies against CSF-1 do not inhibit IL-34 binding to CSF-1R, and antibodies against IL-34 do not inhibit CSF-1 binding to CSF-1R. Ligand binding to the receptor may affect cancer growth. In addition, antibodies to the ligand are not internalized, and they do not induce CSF-IR degradation nor stimulate ADCC activity on cells.

There is a need for a multifunctional antibody that blocks the binding of the ligand to CSF-IR and also induces ADCC activity on cells bound by the antibody.

The antibodies of the present invention are advantageous over known antibodies because they have multiple functions. The antibodies of the present invention block CSF-1 and IL-34 binding to receptors that function to prevent macrophage-induced tumor growth, thereby preventing dimerization of the receptor and thereby phosphorylation of intracellular tyrosine residues. The antibodies of the invention are internalized and induce CSF-IR degradation. Importantly, the antibodies of the present invention enhance ADCC activity by stimulating the death of tumor cells and tumor-associated macrophages and monocytes, as well as ligand binding inhibition. The antibodies of the present invention also simultaneously affect macrophage activity, which plays a major role in tumor progression. The antibodies of the present invention have significant advantages over antibodies known in the art due to their many therapeutic functions.

SUMMARY OF THE INVENTION [

One aspect of the invention is a CDRH1 comprising the sequence SYGMH (SEQ ID NO: 1), a CDRH2 comprising the sequence VIWYDGSNKYYADSVKG (SEQ ID NO: 2), a CDRH3 comprising the sequence GDYEVDYGMDV (SEQ ID NO: 3), a CDRL1 comprising the sequence RASQGISNALA 1R variant (SEQ ID NO: 15) comprising CDRL2 comprising the sequence DASSLES (SEQ ID NO: 5), and CDRL3 comprising the sequence QQFNSYPWT (SEQ ID NO: 6), or a fragment thereof. Another aspect of the invention is a CDRH1 comprising the sequence SYGMH (SEQ ID NO: 1), a CDRH2 comprising the sequence VIWYDGSNKYYADSVKG (SEQ ID NO: 2), a CDRH3 comprising the sequence GDYEVDYGMDV (SEQ ID NO: 3), a CDRL1 comprising the sequence RASQGISNALA , CDRL2 comprising the sequence DASSLES (SEQ ID NO: 5), and CDRL3 comprising the sequence QQFNSYPWT (SEQ ID NO: 6), or a fragment thereof that specifically binds human CSF-1R (SEQ ID NO: 16). One aspect of the invention is a CDRH1 comprising the sequence SYGMH (SEQ ID NO: 1), a CDRH2 comprising the sequence VIWYDGSNKYYADSVKG (SEQ ID NO: 2), a CDRH3 comprising the sequence GDYEVDYGMDV (SEQ ID NO: 3), a CDRL1 comprising the sequence RASQGISNALA (SEQ ID NO: 1R (SEQ ID NO: 15) comprising CDRL2 comprising the sequence DASSLES (SEQ ID NO: 5) and CDRL3 comprising the sequence QQFNSYPWT (SEQ ID NO: 6), or fragments thereof. An antibody of the invention may further comprise an amino acid substitution within one of the CDR sequences. In yet another aspect, the above-mentioned CDR does not have an amino acid substitution in one of the CDR sequences.

Another aspect of the invention relates to a polynucleotide that binds to CSF-1R and has an amino acid sequence:

VH, including QDQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGEGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYEVDYGMDVWGQGTTVTVAS (SEQ ID NO: 7)

Or amino acid sequence:

AIQLTQSPSSLSASVGDRVTITCRASQGISNALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPWTFGQGTKVEIK (SEQ ID NO: 8)

Or a fragment thereof.

Another aspect of the present invention is an antibody or fragment thereof comprising a light chain comprising the amino acid sequence of SEQ ID NO: 10 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, which binds to CSF-1R. In yet another aspect of the invention, the antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 10 and two heavy chains each comprising the amino acid sequence of SEQ ID NO: 9.

CSF-1R-binding fragments of such antibodies are part of the present invention.

The present invention also provides isolated DNA and polynucleotide / poly-nucleic acid encoding the antibody or fragments thereof described above, an expression vector comprising said polynucleotide, and a host cell comprising said polynucleotide. The present invention further provides a method of purifying an antibody or fragment thereof. The invention further provides a pharmaceutical composition comprising an antibody of the invention or a fragment, polynucleotide, vector or host cell thereof alone or in combination with a pharmaceutically acceptable carrier, diluent or excipient. The invention provides a pharmaceutical composition comprising an antibody or fragment thereof of the invention together with a pharmaceutically acceptable carrier, diluent or excipient.

In addition, the present invention relates to a method of inhibiting the growth of cancer cells in a mammal, and a method of treating leukemia, breast and prostate carcinoma, by administering an effective amount of the antibody. Another aspect of the invention relates to a method of inhibiting the growth of cancer cells in a mammal, and a method of treating leukemia, endometrial, breast and prostate carcinoma, by administering an effective amount of the antibody. Another aspect of the present invention is a method of inhibiting the growth of cancer cells and a method of treating ovarian cancer, colorectal cancer, hepatocellular carcinoma, renal cancer, multiple myeloma and Hodgkin's lymphoma as well as leukemia, endometrial, breast and prostate carcinoma . The antibodies of the present invention may be used for the treatment of neoplastic disease (including solid tumors), and for the treatment of breast and prostate carcinomas. In another aspect, the antibodies of the invention may be used for the treatment of neoplastic disease (including solid tumors), and for the treatment of breast, endometrial and prostate carcinomas. In another aspect, the antibodies of the invention may be used for the treatment of neoplastic disease (including solid tumors) and for the treatment of breast, endometrial, prostate, ovarian, colon, rectum, hepatic and renal carcinomas.

One aspect of the invention is an antibody or fragment thereof for use in therapy, or for use in therapy, or for use as a medicament. In yet another aspect, the antibody or fragments thereof described hereinbefore are for use in the treatment of cancer. The present invention may be used to treat cancers including, but not limited to, leukemia, breast cancer, and prostate cancer. In one aspect, the invention can be used to treat cancers including, but not limited to, leukemia, breast cancer, endometrial cancer, and prostate cancer. In another aspect, the invention can be used to treat cancers including, but not limited to, leukemia, breast cancer, endometrial cancer, prostate cancer, ovarian cancer, colorectal cancer, hepatocellular carcinoma, renal cancer, multiple myeloma and Hodgkin's lymphoma .

The present invention also encompasses an antibody or fragment thereof of the present invention for use in the treatment of cancer comprising the administration or administration of an effective amount of another anti-cancer therapeutic agent, wherein the anti-cancer therapeutic agent is an anti-angiogenic agent, But are not limited to. In addition, the antineoplastic agents is not limited to one including docetaxel, paclitaxel, Herceptin (Herceptin) ^® and doxorubicin. The antibody or fragment disclosed herein as well as the anti-cancer therapeutic agent are administered to the patient. The antibody or fragment thereof is administered before, during, substantially simultaneously with, or after the start of a therapy using another anti-cancer therapeutic agent or another anti-neoplastic agent.

The invention also provides the use of an antibody of the invention for the manufacture of a medicament for the treatment of cancer. In one aspect, the cancer is leukemia, breast cancer, or prostate cancer. In one aspect, the cancer is leukemia, breast cancer, endometrial cancer, or prostate cancer. In another aspect of the invention, the cancer is leukemia, breast cancer, endometrial cancer, prostate cancer, ovarian cancer, colorectal cancer, hepatocellular carcinoma, renal cancer, multiple myeloma or Hodgkin's lymphoma. The use of the antibody includes the administration or administration of an effective amount of another anti-cancer therapeutic agent, wherein the anti-cancer therapeutic agent includes, but is not limited to, an anti-angiogenic agent, a chemotherapeutic agent or an anti-neoplastic agent. In addition, the antineoplastic agents is not limited to one including docetaxel, paclitaxel, doxorubicin and Herceptin ^®. The antibody or fragment disclosed herein as well as the anti-cancer therapeutic agent are administered to the patient. The antibody or fragment thereof is administered before, during, substantially simultaneously with, or after the start of a therapy using another anti-cancer therapeutic agent or another anti-neoplastic agent.

The present invention further provides a method of treating cancer in a mammal in need thereof, comprising administering to the mammal an effective amount of an antibody or fragment thereof of the invention. Cancer is selected from the group consisting of leukemia, breast cancer, endometrial cancer and prostate cancer. In another aspect, the cancer is selected from the group consisting of leukemia, breast cancer, endometrial cancer, prostate cancer, ovarian cancer, colorectal cancer, hepatocellular carcinoma, renal cancer, pancreatic cancer, multiple myeloma and Hodgkin's lymphoma. In addition, the method may further comprise administering another anti-cancer therapeutic agent to said mammal. The anti-cancer therapeutic agent is selected from the group consisting of an anti-angiogenic agent, a chemotherapeutic agent, and an anti-neoplastic agent. Antineoplastic agents may be selected from the group consisting of docetaxel, paclitaxel, doxorubicin and Herceptin ^®.

The present invention further provides a method of using an antibody or composition for the treatment of a mammal in need thereof, such as inhibition of angiogenesis or bone metastasis, or inhibition of tumor or hyperplastic growth, or treatment of inflammatory diseases do. The present invention further provides an antibody or composition for use in the treatment of a mammal in need thereof, such as inhibition of angiogenesis or bone metastasis, or inhibition of tumor or hyperplastic growth, or treatment of inflammatory diseases.

The invention also relates to a product or pharmaceutical composition containing the antibody or fragments thereof disclosed herein. In addition, the product or the pharmaceutical composition at the same time as the antibodies disclosed herein in therapy, individually or more of the pharmaceutical agents that are sequentially or in combination with an antineoplastic agent or an anticancer agent or a therapeutic agent (docetaxel, paclitaxel, Herceptin ^®, or include, doxorubicin limited (Not shown).

The present invention relates to the use of a CSF-1 level in a sample of blood, serum, plasma, tumor cells and circulating tumor cells in a patient in whom the cancer has CSF-1R expressed on the surface of tumor-associated macrophages, RTI ID = 0.0 > 1R < / RTI > antibody or fragment thereof. The present invention also relates to a method of treating a patient suffering from (1) measuring the level of CSF-1 in a sample from a patient selected from the group consisting of blood, serum, plasma, tumor cells and circulating tumor cells, and (2) Comprising the step of administering to the patient an antibody or fragment thereof of the invention when the level of CSF-1 is greater than the level of CSF-1 found in the patient.

The present invention provides for the use of IL-34 levels in samples of blood, serum, plasma, tumor cells and circulating tumor cells as indicators of successful treatment of patients with the CSF-1R antibodies or fragments thereof of the invention in a patient. The present invention also relates to a method of treating a subject suffering from (1) measuring the level of IL-34 in a sample from a patient selected from the group consisting of blood, serum, plasma, tumor cells and circulating tumor cells, and (2) Comprising administering to the patient an antibody or fragment thereof of the invention when the level of IL-34 is greater than the level of IL-34 found in the patient.

One aspect of the present invention comprises (1) determining the level of CSF-1 in a sample of a subject having cancer in vitro or in vitro, wherein the sample is a blood, serum, plasma, tumor cells, and circulating tumor cells ≪ / RTI > (2) an increase in the level of CSF-1 relative to the level of CSF-1 in the non-cancerous individual indicates that the subject is a candidate for anti-CSF-1R antibody-based cancer therapy, It is a method of determining whether a subject having an cancer, an antibody of the invention, is a candidate for anti-CSF-1R antibody-based cancer therapy.

Another aspect of the present invention comprises (1) determining the level of IL-34 in a sample of a subject having an cancer, either ex vivo or in vitro, wherein the sample comprises blood, serum, plasma, tumor cells, and circulating tumor cells ≪ / RTI > (2) an increase in the level of IL-34 compared to the level of IL-34 in the non-cancerous individual indicates that the subject is a candidate for anti-CSF-1R antibody-based cancer therapies, It is a method of determining whether a subject having an cancer, an antibody of the invention, is a candidate for anti-CSF-1R antibody-based cancer therapy.

The present invention also provides an antibody that specifically binds to CSF-1R. (A) inhibition of binding of CSF-1 or IL-34 to CSF-1R; (b) inhibiting activation of CSF-1R; (c) reduction of phosphorylation of CSF-1R; (d) reduced activation of MAPK; (e) reduced activation of Akt; (f) reduction in the amount of CSF-1R; And (g) ADCC induction. Preferred embodiments of the present invention have the characteristics (a) to (g).

One aspect of the invention is directed to a method of inhibiting the signaling of CSF-1R, in particular CSF-1R (SEQ ID NO: 15) or human CSF-1R , An antibody or a fragment thereof that stimulates antibody-dependent cell-mediated cytotoxicity (ADCC) activity on various cells including tumors, macrophages and monocytes. SEQ ID NO: 15 and SEQ ID NO: 16 differ in one amino acid at position 54 outside the binding region.

As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains of two heavy chains (H) and two light chains (L) linked by a disulfide bond. Individual chains can be folded into domains of size (110-125 amino acids) and domains that are similar in structure but different in function.

The light chain may comprise one variable domain (abbreviated herein as VL) and / or one constant domain (abbreviated herein as CL). The light chain of a human antibody (immunoglobulin) is kappa (K) light chain or lambda (lambda) light chain. As used herein, the expression VL comprises both a variable region of a kappa-type light chain (VK) and a variable region of a lambda-type light chain (V 貫). Also, the heavy chain may comprise three or four constant domains (CH1, CH2, CH3 and CH4) (collectively referred to herein as CH), depending on the class or isotype of the antibody and / or one variable domain (abbreviated herein as VH) ). In humans, the isoforms are IgA, IgD, IgE, IgG and IgM, and IgA and IgG are further subdivided into subclasses or subtypes (IgA1-2 and IgG1-4). The present invention includes antibodies of any of the above-mentioned classes or subclasses. The human IgG ₁ isotype are preferred as the antibodies of the invention.

Three regions, called hypervariable or complementarity-determining regions (CDRs), are found in each of the VL and VH, which is supported by a less variable region called a framework (abbreviated herein as FR). Amino acids can be prepared by methods known in the art including, but not limited to, Kabat custom (Kabat, et al., Ann. NY Acad. Sci. 190: 382-93 (1971); Kabat, et al., Sequences of Proteins of Immunological Interest, (1991)), which is assigned to a particular CDR region or domain. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino-terminus to the carboxy-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Some of the antibodies consisting of the VL and VH domains are designated Fv (variable fragment) and constitute antigen-binding sites. Single chain Fv (scFv) is an antibody fragment containing a VL domain and a VH domain on one polypeptide chain, wherein the N terminus of one domain and the C terminus of the other domain are linked by a soluble linker.

"Isolated antibody" refers to an antibody that is partially, substantially or completely purified from a mixture of (1) components, (2) an antibody identified and isolated and / or recovered from a component of the natural environment, (3) a monoclonal antibody, (4) an antibody that does not contain other proteins from the same species, (5) an antibody that is expressed by cells of a different species, or (6) an antibody that does not occur in nature. The components exclude antibodies of the invention, as used herein. Contaminant components of the natural environment of the antibody are substances that interfere with the diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. Examples of isolated antibodies include affinity purified antibodies, antibodies prepared by in vitro hybridoma or other cell lines, and human antibodies derived from transgenic mice.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, for example, individual antibodies that make up a population may contain possible naturally occurring mutations or minority translations Are substantially the same except for the post mutation. Monoclonal antibodies are highly specific and are directed against a single antigenic site (also known as a crystallizer or epitope). In addition, in contrast to conventional (polyclonal) antibody preparations, which typically comprise different antibodies directed against different determinants, each monoclonal antibody is directed against the monoclonal antibody on the antigen. The modifier "monoclonal" refers to the characteristics of an antibody as being obtained from a population of substantially homogeneous antibodies, and should not be construed as requiring production of the antibody by any particular method.

As used herein, the term "human antibody" encompasses antibodies having variable and constant regions corresponding to the human intervening immunoglobulin sequences (as described in Kabat et al., Supra). Human antibodies of the present invention can be modified by, for example, introducing into the CDR an amino acid residue that is not encoded by the human guinea pig immunoglobulin sequence (e. G., Introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) Lt; / RTI > mutants). Human antibodies can be replaced with amino acid residues that are not coded for by the human wiring immunoglobulin sequence in at least one position, e. G., An active enhancing amino acid residue. However, as used herein, the term "human antibody" does not include antibodies that have been transplanted into a human framework sequence with a CDR sequence derived from another mammalian species, e. As used herein, the methods of producing "human antibodies" do not include antibodies produced in humans.

The phrase "recombinant human antibody" is intended to encompass a human antibody produced, expressed, produced or isolated by recombinant means, such as an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant human antibody library, An antibody isolated from an animal transgenic against an immunoglobulin gene or an antibody produced, expressed, produced or isolated by any other means, including splicing a human immunoglobulin gene sequence to another DNA sequence . Such a recombinant human antibody has a variable region and a constant region derived from a human wiring immunoglobulin sequence.

Fc (fragment, crystallizable region) is a name for a part or fragment of an antibody composed of paired heavy chain constant domains. For example, in an IgG antibody, Fc consists of the heavy chain CH2 and CH3 domains. The Fc of an IgA or IgM antibody further comprises a CH4 domain. Fc is associated with activation of Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC) and / or cell mediated cytotoxicity (CMC). In the case of antibodies that are multiple IgG-like protein complexes, such as IgA and IgM, Fc constant domains are required for complex formation.

Thus, an antibody of the invention can be used in combination with an isolated antibody, a human antibody, a humanized antibody, a recombinant human antibody, a monoclonal antibody, a digestion fragment, a specific portion and variants thereof (including antibody mimetics, Including, but not limited to, a portion of an antibody that mimics the structure and / or function of the antibody, each containing at least one CDR. The functional fragment comprises an antigen-binding fragment that binds to a CSF-1R antigen. For example, an antibody fragment that can bind to CSF-1R or a portion thereof and is included in the present invention is a bivalent fragment, such as a Fab (Fab ') ₂ , monovalent fragment having an intact interchain disulfide bond, such as VL- Fab (fragment, antigen binding) that refers to a fragment of an antibody consisting of CL and VL-CHl domains and does not have a heavy chain hinge region (e.g., by papain digestion), Fab having a heavy chain hinge region, Facb F (ab ') ₂ , Fab' lacking a disulfide bond, pFc '(for example, by pepsin or plasmin digestion), Fd (for example, by pepsin Digestion, partial reduction and re-aggregation), and Fv or scFv (e. G., By molecular biology techniques). In addition, the antibody fragment may be, for example, a domain specific antibody, a linear antibody, a single chain antibody, a scFv, a single domain antibody, a polyvalent single chain antibody, a multispecific antibody formed from antibody fragments, , Triabodies, and the like.

The hinge region separates the Fab and Fc portions of the antibody not only to provide mobility for Fab and Fc for Fab, but also to include multiple disulfide bonds for covalent linkage of the two heavy chains.

An antibody format that retains binding specificity but has other characteristics that may be desirable, including, for example, bispecific, multivalent (more than two binding sites) and miniaturized (e.g., binding domain alone) Has been developed. The antibodies of the present invention are specific for CSF-1R. Antibody specificity refers to the selective recognition of an antibody to a particular epitope of an antigen. The antibodies of the present invention can be, for example, monospecific or bispecific. A bispecific antibody (BsAb) is an antibody having two different antigen-binding specificities or sites. An epitope recognized when the antibody has more than one specificity may be associated with a single antigen or more than one antigen. Thus, the present invention provides bispecific antibodies that bind to two different antigens with at least one specificity for CSF-1R. As mentioned above, such antibodies include any fragment thereof.

The specificity of the antibody or fragment thereof of the present invention for CSF-1R can be determined based on affinity and / or binding ability. The affinity is expressed as the equilibrium constant (K _D ) for dissociation of the antibody and antigen, and measures the binding strength between the antigenic determinant and the antibody-binding site.

The antibody or fragment thereof of the invention binds to an epitope of CSF-1R located on an extracellular domain segment (hereinafter simply referred to as "domain" or "ECD"). As used herein, the term "epitope" refers to a discontinuous three-dimensional region of an antigenic phase recognized by an antibody of the invention. An epitope is an immunologically active region of a complex antigen that is actually a region that binds to the B-cell receptor and is bound by antibody molecules produced by B cells. Generally, the antigen contains at least one epitope, typically more than one epitope. The epitope on the protein antigen may be linear or non-linear. A linear epitope is an epitope composed of contiguous amino acid residues of a protein amino acid sequence. Linear epitopes may or may not require morphological folding in order to elicit an immune response that produces antibodies that form natural three-dimensional structures and binding specificities to the antigen. Non-linear epitopes consist of non-contiguous amino acid residues. Thus, non-linear epitopes are always required to have some degree of protein folding that approximates the amino acid residues necessary to elicit an immune response to form a natural three-dimensional structure and to fish an antibody that has binding specificity for the antigen.

The antibodies of the present invention also include improved binding properties by direct mutagenesis, affinity maturation, phage display, or by methods of chain shuffling. Affinity and specificity can be modified or improved by mutating the CDR and / or FW residues and screening for antigen binding sites with desirable properties (see, e.g., Yang et al., J. Mol. Biol. , 254: 392-403 (1995)). CDRs are mutated in a variety of ways. One method is to randomize individual residues or combinations of residues so that a subset of 2 to 20 amino acids is found in a particular location within a population of other identical antigen binding sites. Alternatively, mutagenesis can be induced over a range of residues by error-prone PCR methods (see, e.g., Hawkins et al., J. Mol. Biol., 226: 889-96 (1992) Reference). In another example, a phage display vector containing heavy and light chain variable region genes is shown. (See, for example, Low et al., J. Mol. Biol., 250: 359-68 (1996)) in E. coli mutagenesis strains. Such mutagenesis methods are examples of many methods known to those skilled in the art.

A convenient method of generating substitutional variants is affinity maturation using phage display. Briefly, some CDR region regions are mutated to produce all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a univariate fashion from the filamentous phage particle as a fusion to the gene III product of M13 packaged in each particle. The phage-displayed mutants are then screened for their biological activity (e. G., Binding affinity (K _D ), specificity, EC50) as described herein. To identify candidate CDR region sites for transformation, alanine scanning mutagenesis can be performed to identify CDR region residues that contribute significantly to antigen binding. Alternatively or additionally, random mutagenesis can be performed at one or more residue positions of one or more CDR sequences, regardless of whether the CDR is operably linked to the variable region or the CDR is independent of other variable region sequences And then the altered CDR is returned to the variable region using recombinant DNA technology. Once such variant antibodies are generated and expressed, panels of variants can be screened as described herein and antibodies with superior characteristics in one or more related assays can be selected for further development.

As well as the antibodies specifically described herein, other "substantially homologous " modified antibodies can be readily designed and constructed using a variety of recombinant DNA techniques known to those skilled in the art. For example, framework regions may differ from native sequences at the primary structural level by some amino acid substitution, terminal and intermediate addition and deletion, and the like. In addition, a variety of different human framework regions may be used alone or in combination as a basis for the humanized immunoglobulins of the present invention. In general, the modification of a gene can be easily accomplished by a variety of well-known techniques, such as site-directed mutagenesis.

As disclosed herein, the invention encompasses nucleic acid sequences encoding anti-CSF-1R antibody heavy chain comprising any of the VH regions or portions thereof, or any of the VH CDRs, such as any variants thereof. As disclosed herein, the invention also includes nucleic acid molecules that encode an anti-CSF-1R antibody light chain comprising any one of the VL region or portion thereof or a VL CDR, such as any variant thereof. The invention also encompasses nucleic acid sequences of antibody 1, sequences 13 and 14, respectively, for heavy and light chains. An antibody of the invention comprises an antibody comprising the same CDR region of antibody 1, and / or the same light chain variable region and / or heavy chain variable region of antibody 1.

The antibodies of the present invention can be produced by methods known in the art. These methods are described in Kohler and Milstein, Nature 256: 495-497 (1975); Immunologically, as described in " Monoclonal Antibody Technology, < / RTI > Production and Characterization of Rodent and Human Hybridomas (Campbell ed., 1984) by Burdon et al. As well as recombinant DNA methods described in Huse et al., Science 246: 1275-1281 (1989). The antibody may also be obtained from a library that retains a combination of VH and VL domains in scFv or Fab form. The VH and VL domains may be encoded by synthetic, partially synthetic or naturally occurring nucleotides. The invention can be made by a phage display library carrying human antibody fragments. Another source of human antibodies is transgenic mice genetically engineered to express human immunoglobulin genes.

One embodiment for the production of antibodies is to express a nucleic acid encoding an antibody according to the invention in a transgenic animal having a substantial portion of the inserted human antibody-producing genome and resulting in the production of endogenous antibodies. Transgenic animals include, but are not limited to, mice, goats, and rabbits. The antibodies of the invention are made with transgenic mice. One further embodiment of the present invention includes, for example, expressing an antibody-coding gene in the mammary gland such that the polypeptide is secreted during lactation.

Conventional methods for generating "humanized" antibodies include transplanting CDR sequences from MAbs (produced by immunizing a rodent host) into human Ig backbones, and transfecting chimeric genes with Chinese hamster ovary (CHO) cells (Which again produce functional antibodies secreted by CHO cells).

An amino acid residue that is the primary determinant of the binding of a single domain antibody may be present in a Kabat or cotyline defined CDR, but may be present in another, such as a residue that is otherwise embedded in the VH-VL interface of the VH- It is understood that moieties may also be included.

The protein used to identify the CSF-1R binding antibody of the present invention is preferably CSF-1R, more preferably the extracellular domain of CSF-1R. The CSF-1R extracellular domain may not contain another molecule or may be conjugated to another molecule. The antibodies of the present invention may be fused to additional amino acid residues. Such an amino acid residue may be an IgG FC portion to optimize the peptide tag or dimer and which may be to facilitate isolation. Other amino acid residues for targeting antibodies to specific organs or tissues are also contemplated.

Antibody fragments can be produced by digesting intact antibodies or by expressing DNA encoding the fragments. Fragments of antibodies are described in Lamoyi et al., J. Immunol. Methods 56: 235-243 (1983) and Parham, J. Immunol. 131: 2895-2902 (1983). Such fragments may contain one or both of the Fab fragments or the F (ab ') ₂ fragments. Such fragments may also contain single-chain fragment variable region antibodies, i. E. ScFv, diabodies, or other antibody fragments. Methods for producing such functional equivalents are disclosed in European Patent Application Publication No. EP 239,400 (Winter); PCT Publication WO 89/09622 (Hann, et al.); European Patent Application Publication No. EP 338,745 (Owens, et al.); And European Patent Application Publication No. EP 332,424 (Beldler, et al.). Throughout this specification, the term "antibody" of the present invention encompasses any fragment thereof whether or not specifically mentioned.

Preferred host cells for transformation of the vectors of the invention and expression of the antibodies of the invention include mammalian cells such as NS0 cells (non-secreting (0) mouse myeloma cells), 293, SP20 and CHO cells, and lymphomas, Other cell lines of lymphoid origin such as myeloma or hybridoma cells. Other eukaryotic hosts, such as yeast, may alternatively be used.

The antibodies of the present invention may be purified by any method known in the art, for example, precipitation with ammonium sulfate or sodium sulfate followed by dialysis against saline, ion exchange chromatography, affinity or immunoaffinity chromatography as well as gel filtration or Can be isolated or purified by electrophoresis. A preferred purification method for the antibodies of the present invention is protein-A affinity chromatography.

The DNA encoding human antibodies can be prepared by recombinant DNA encoding human constant regions and variable regions other than CDRs and CDRs derived from human, substantially or only from a corresponding human antibody region .

Suitable sources of DNA encoding fragments of antibodies include any cells such as hybridoma and spleen cells expressing full length antibodies. As described above, the fragment may itself be used as an antibody equivalent, or may be recombined into an equivalent. The DNA recombination and other techniques described herein can be performed by known methods. Another source of DNA is the phage display library of antibodies as is known in the art.

In addition, the invention provides expression vectors containing previously described polynucleotide sequences operatively linked to a reference sequence, such as an expression sequence, a promoter and / or an enhancer sequence. A variety of expression vectors have been developed for the efficient synthesis of antibody polypeptides within prokaryotic systems such as bacteria, and eukaryotic systems including, but not limited to, yeast and mammalian cell culture systems. The vector of the present invention may comprise fragments of chromosomal, non-chromosomal and synthetic DNA sequences.

Any suitable expression vector may be used. For example, a prokaryotic cloning vector may be used for this. Plasmids from E. coli such as colEl, pCRl, pBR322, pMB9, pUC, pKSM and RP4. Prokaryotic vectors also include phage DNA, such as M13 and derivatives of other filamentous single-stranded DNA phages. An example of a vector useful in yeast is a 2μ plasmid. Suitable vectors for expression in mammalian cells are derived from SV-40, CMV, adenovirus, known derivatives of retrovirus-derived DNA sequences, and combinations of functional mammalian vectors and functional plasmids and phage DNAs as described above Lt; / RTI > Additional eukaryotic expression vectors are known in the art (see, for example, PJ Southern and P. Berg, J. Mol. Appl. Genet. 1: 327-41 (1982); Subramani et al., Mol. (1982); Scahill et al., Proc. Nat'l Acad. Sci. USA 80: 4654 (1981); Kaufmann and Sharp, J. Mol. Biol. 15: Urlaub and Chasin, Proc Nat'l Acad Sci USA 77: 4216-20 (1980)).

An expression vector useful in the present invention contains at least one expression control sequence operably linked to a DNA sequence or fragment to be expressed. The control sequences are inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the major operator and promoter region of phage lambda, the regulatory region of the fd coat protein, the glycosylation promoter of yeast such as 3-phosphoglycerate Promoters for kinases, promoters of yeast acid phosphatase, such as Pho5, promoters of yeast alpha-splicing factors, and promoters derived from polyomas, adenoviruses, retroviruses and monkey viruses, such as early and late promoters or SV40 , And other sequences or combinations thereof known to control the gene expression of prokaryotic or eukaryotic cells and their viruses.

When it is desirable to express a gene construct in yeast, a suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7. The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, e. g., ATCC No. 44076. The presence of trp1 lesions in the yeast host cell genome then provides an effective environment for detecting transformation by growing in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids carrying the Leu2 gene.

The present invention also provides recombinant host cells containing the recombinant vectors described previously. The antibody of the present invention can be expressed in a cell line other than the hybridoma. Nucleic acid containing a sequence encoding a polypeptide according to the present invention may be used for transformation of suitable mammalian host cells.

Particularly preferred cell lines are selected based on high levels of expression, constitutive expression of the protein of interest, and minimal contamination from the host protein. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines such as, but not limited to, COS-7 cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) And many other cell lines, including those of lymphoid origin, such as lymphoma, myeloma or hybridoma cells. Suitable additional eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, e. Coli, e. Coli SG-936, i. Coli HB 101, < / RTI > E. coli W3110, i. Coli X1776, i. Coli X2282, i. Coli DHI, and < RTI ID = 0.0 > Coli MRCl, and a Pseudomonas (Pseudomonas), Bacillus (Bacillus), for example Bacillus subtilis (Bacillus subtilis) and streptomycin MRS (Streptomyces).

Such recombinant host cells can be used to produce antibodies or fragments thereof by culturing the cells under conditions permitting expression of the antibody or fragment thereof and purifying the antibody or fragment thereof from the host cell or a medium around the host cell. Targeting of the expressed antibody or fragment for secretion in the recombinant host cell may be facilitated by inserting a signal or secretory leader peptide-coding sequence at the 5 ' end of the antibody-coding gene of interest. Such secretory leader peptide elements can be derived from prokaryotic or eukaryotic sequences. Thus, a suitable secretory leader peptide is used that is an amino acid linked to the N-terminal end of the polypeptide, directing the polypeptide to travel outside the host cell cytosol and secreted into the medium.

The transformed host cells can be transformed with a suitable carrier such as carbon (carbohydrates such as glucose or lactose), nitrogen (amino acids, peptides, proteins or their degradation products such as peptones, ammonium salts, etc.) Potassium, magnesium and calcium sulphate, phosphate and / or carbonate) in a liquid medium containing an assimilable source. The medium additionally contains, for example, growth-promoting substances such as trace elements such as iron, zinc, manganese and the like.

The present invention also provides a method of treating tumor growth in a mammal by administering to the mammal an effective amount of the antibody. Suitable conditions for treatment according to the present invention preferably involve cells expressing CSF-1R. Without wishing to be bound by any particular mechanism, the methods of the present invention may be used for the treatment of immune disorders such as, for example, cancer cell growth including neoplastic growth, bone metastasis, rejection of organ transplantation or autoimmune diseases stimulated by CSF-IR ≪ / RTI >

In the context of the present invention, "treating" or "treating" refers to inhibiting, slowing, reducing or reversing the progression of a fundamental or unwanted physiological change associated with a disease or disorder, Including the prevention of the onset of clinical symptoms of the disease. A beneficial or desired clinical result may include a reduction in symptoms, a reduction in the severity of the disease or disorder, a stabilization of the disease or disorder (i. E., The disease or disorder is not worsened), a delay in disease or disorder progression, Slowing, alleviation or amelioration of the disease or disorder, and improvement of the disease or disorder (whether partial or total). Treatment may also mean prolonging survival as compared to expected survival in the absence of treatment. Those in need of treatment include those already with the disease. In one embodiment, the invention can be used as a medicament.

In the methods of the invention, a therapeutically effective amount of an antibody of the invention is administered to a mammal or patient in need thereof. In addition, the pharmaceutical compositions of the present invention may comprise a therapeutically effective amount of an anti-CSF-1R antibody of the invention. The "therapeutically effective amount" or "effective dose" refers to an amount that is effective for achieving the desired therapeutic result and an amount effective for the period of time. The therapeutically effective amount of the antibody may vary depending on factors such as, for example, the disease state of the individual, age, sex, and body weight, and the ability of the antibody or antibody portion to elicit a desired response in the individual. Other factors include the administration, the target site, the physiological condition of the patient (whether the patient is a human being or an animal), the other medication being administered, and whether the treatment is prophylactic or therapeutic. The human antibodies of the present invention are particularly useful for administration to humans, but may also be administered to other mammals. As used herein, the term mammal includes, but is not limited to, humans, laboratory animals, domestic pets and livestock animals. In addition, a therapeutically effective amount is a greater amount of the therapeutically beneficial effect than any toxic or deleterious effect of the antibody or antibody portion.

The dosage regimen may be adjusted to provide a desired optimal response (e. G., A therapeutic or prophylactic response). Therapeutic dosages can be tailored to optimize safety and efficacy using conventional methods known to those skilled in the art. Typically, the dosing schedule will range from single bolus administration or continuous infusion to several doses per day (e. G., Every 4-6 hours), or as directed by the therapist and the condition of the patient. An exemplary non-limiting range for a therapeutically effective amount of an antibody of the invention is 0.1-50 mg / kg, more preferably 3-35 mg / kg, and more preferably 5-20 mg / kg. Dosage and frequency of administration will vary from less than 1 mg / kg to greater than 100 mg / kg, as provided by the physician treating the patient and provided daily, three times per week, once every two weeks, or less frequently Capacity. It should be noted, however, that the present invention is not limited to any particular capacity.

Anti-CSF-1R antibodies can be administered in combination with one or more other chemotherapeutic therapies including, but not limited to, antiangiogenic, chemotherapeutic, and anti-neoplastic agents. Any suitable anti-cancer agent, such as a chemotherapeutic agent, radiation, an antibody, or a combination thereof, may be used.

Anticancer agents include, but are not limited to, anti-neoplastic agents, antibodies, azants and prodrugs. Antineobiological agents that are currently known or appreciated in the art can be used in a wide variety of classes such as mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, A survival agent, a biological response modifier, an anti-hormone agent, and an anti-angiogenic agent. Examples of alkylating agents include, but are not limited to, cisplatin, cyclophosphamide, melphalan and saccharin. Examples of water metabolism, wherein the doxorubicin, daunorubicin, paclitaxel, gemcitabine, alrimta (ALIMTA) ^® and topoisomerase inhibitors irinotecan (CPT-11), amino camptothecin, camptothecin, DX-8951f, topotecan ( (Topoisomerase I), etoposide (VP-16), and tenifocide (VM-26) (topoisomerase II). If the anti-neoplastic agent is radiation, the source of the radiation may be external (external beam radiation therapy - EBRT) or internal (proximal therapy - BT) for the patient to be treated. The dose of the anti-neoplastic agent administered will depend on a number of factors including, for example, the type of agent, the type and severity of the tumor to be treated, and the route of administration of the agent. It should be emphasized, however, that the present invention is not limited to any particular capacity. In one aspect, docetaxel is the preferred anti-neoplastic agent of the present invention. In another aspect of the invention, paclitaxel, Herceptin ( ^R) and doxorubicin are preferred anti-neoplastic agents.

The anti-CSF-1R antibodies of the invention can be administered with antibodies that neutralize other receptors involved in tumor growth or angiogenesis. In one embodiment of the invention, the anti-CSF-1R antibody is used in combination with a receptor antagonist that specifically binds Her2. Another example of such a receptor is VEGFR. Anti-CSF-1R antibodies can be used in combination with VEGFR antagonists. CSF-1R antibodies can be administered in combination with one or more suitable adjuvants such as cytokines (e.g., IL-10, IL-4 and IL-13) or other immunostimulatory agents such as (but not limited to) chemokines, May be administered in combination with related antigens and peptides.

In the present invention, any suitable method or route may be used to administer the anti-CSF-1R antibodies of the invention, optionally co-administer antineoplastic agents and / or antagonists of other receptors. In the combination therapy of the present invention, wherein -CSF-1R antibody is docetaxel, paclitaxel, doxorubicin or Herceptin ^® as well as before the beginning of the therapy, including their use any combination of the other agents are not limited thereto, and for , Substantially simultaneously with, or after the administration of the compound. Anti-neoplastic agents used in accordance with the present invention include any therapy that is considered optimal for the treatment of a neoplastic condition in a patient. Several malignant tumors may require the use of specific anti-tumor antibodies and specific anti-neoplastic agents that will be determined differently depending on the patient. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration. The dosage of the antagonist to be administered will depend on a number of factors including, for example, the type of antagonist, the type and severity of the tumor to be treated, and the route of administration of the antagonist. It should be emphasized, however, that the present invention is not limited to any particular method or route of administration.

The anti-CSF-1R antibodies of the present invention are preferably formulated into pharmaceutical compositions when used in mammals for the purpose of prevention or treatment. Such pharmaceutical compositions and methods for their preparation are well known in the art. For example, the literature: see [Remenigton The Science and Practice of Pharmacy (A. Gennaro, et al, eds, 19 ^th ed, Mack Publishing Co., 1995...)].

In another aspect of the invention, an antibody of the invention may be administered, or chemically or biosynthetically linked, with an anti-cancer agent, an anti-neoplastic agent, or an anti-angiogenic agent or a detectable signal-generating agent. An antitumor agent linked to an antibody includes any agonist that destroys or damages the neoplastic vasculature to which the antibody is bound or the environment of the tumor or macrophage or cell to which the antibody is bound. The present invention may be an anti-CSF-1R antibody that specifically binds to a receptor and is administered as a conjugate including, but not limited to, an immunoconjugate that delivers a toxin upon ligand-toxin internalization. The antibody-agonist conjugates can be linked directly to each other or through linkers, peptides or non-peptides. For example, the antitumor agent or anti-macrophage agent is a toxic agent such as an anti-neoplastic agent or a radioactive isotope. Suitable anti-neoplastic agents, including chemotherapeutic agents, are well known to those skilled in the art and are discussed below. The present invention further relates to a method for the detection of an anti-CSF-antibody that is linked to a target or reporter moiety comprising, for example, an anti-neoplastic agent, another antibody or a reporter such as a radioactive isotope, in a diagnostic system in which the detectable signal- 1R < / RTI >

In accordance with the present invention, a method of inhibiting angiogenesis comprises administering to a mammal a composition comprising an antibody or antibody fragment of the invention for a period of time and in an amount effective to inhibit angiogenesis. Similarly, antibodies and antibody fragments can be used in a method for inhibiting tumor metastasis in a mammal by administering to the mammal an amount of the antibody of the invention that is effective to inhibit metastasis for a period of time.

In accordance with the present invention, a method of inhibiting macrophage infiltration into a tumor matrix and stimulating tumor growth comprises administering to the mammal an amount of the antibody or antibody of the invention that is effective to inhibit the effect of macrophages on the growth and tumor growth for a period of time and / Comprising administering a composition comprising an antibody fragment. Similarly, antibodies and antibody fragments may be used in a method for alleviating bone erosion around a tumor metastasis in a mammal by administering to the mammal an amount of the antibody of the invention that is effective to inhibit bone erosion for a period of time. have.

The present invention relates to the use of a CSF-1R ligand as a biomarker for a cancer patient responsive to treatment when the cancer has CSF-1R expressed on the surface of tumor-associated macrophages in blood, serum, plasma, tumor cells or circulating tumor cells CSF-1 or IL-34). The present invention further contemplates a method of predicting successful treatment of a patient with an antibody or fragment of the invention by measuring serum CSF-1 levels in a sample selected from the group consisting of serum, plasma, tumor cells or circulating tumor cells. Yet another aspect of the present invention is a method of treating a patient suffering from (1) measuring the level of CSF-1 in a sample from a patient selected from the group consisting of blood, serum, plasma, tumor cells and circulating tumor cells, and (2) Comprising administering to the patient an antibody or fragment thereof of the invention when the level of CSF-1 is greater than the level of CSF-1 found in the control population. The present invention further contemplates a method for predicting successful treatment of a patient with an antibody or fragment of the invention by measuring blood IL-34 levels in a sample selected from the group consisting of serum, plasma, tumor cells or circulating tumor cells. Yet another aspect of the present invention is a method for detecting IL-34 in a sample obtained from a patient selected from the group consisting of (1) blood, serum, plasma, tumor cells and circulating tumor cells, and (2) Comprising administering to the patient an antibody or fragment thereof of the invention when the level of IL-34 found in the control population is higher than the level of IL-34 found in the control population.

(1) determining the level of CSF-1 in a sample of a subject having cancer, either ex vivo or in vitro, wherein the sample comprises blood, serum, plasma, tumor cells, and circulating tumor cells ≪ / RTI > (2) an increase in the level of CSF-1 relative to the level of CSF-1 in the non-cancerous individual indicates that the subject is a candidate for anti-CSF-1R antibody-based cancer therapy, Consider a method of determining whether a subject having an cancer, an antibody of the invention, is a candidate for anti-CSF-1R antibody-based cancer therapy.

(1) determining the level of IL-34 in a sample of a subject having cancer, either ex vivo or in vitro, wherein the sample comprises blood, serum, plasma, tumor cells, and circulating tumor cells ≪ / RTI > (2) an increase in the level of IL-34 compared to the level of IL-34 in the non-cancerous individual indicates that the subject is a candidate for anti-CSF-1R antibody-based cancer therapies, Consider a method of determining whether a subject having an cancer, an antibody of the invention, is a candidate for anti-CSF-1R antibody-based cancer therapy.

The present invention also provides a method of treating cancer, comprising: (1) determining the level of CSF-1 or IL-34 or both in vitro or in vitro in a sample of a subject having cancer, wherein the sample is blood, serum, plasma, And circulating tumor cells; (2) an increase in the level of CSF-1 or IL-34 or both relative to the level of CSF-1 or IL-34 or both in the non-cancerous individual indicates that the subject is anti-CSF- Wherein the antibody is a candidate for cancer therapy, wherein the antibody is a human anti-CSF-1R antibody-based cancer therapeutic regimen, wherein the antibody is an antibody of the invention.

Levels of CSF-1 or IL-34 include commercially available kits (R & D Systems for CSF-1 and USCN Life Sciences, Inc. for IL-34) And can be measured in various ways. In one such technique, a CSF-1 or IL-34 standard or sample is added to a precoated plate as an antibody against human CSF-1 or IL-34 to allow binding of CSF-1 or IL-34 to the antibody do. After washing unbound CSF-1 or IL-34 and other proteins, a secondary antibody recognizing anti-CSF-1 or IL-34 antibody, respectively, is added. The secondary antibody is coupled to horseradish peroxidase which releases a blue light color when added to the well in the substrate. The intensity of the color is related to the amount of CSF-1 or IL-34 found in the plate.

Since CSF-1 and IL-34 occur naturally in healthy human bodies, CSF-1 and IL-34 baselines will need to be determined in the control population. The control population may include a group of individuals that are not diagnosed with a cancerous condition or an indication of infection. Thus, the control group will establish normal or baseline CSF-1 and IL-34 levels for healthy individuals. For example, CSF-1 levels are 68% or 77% higher in breast cancer or colorectal cancer patients than in controls, respectively (Lawicki et al., Clin. Chim. Acta 317: 112-116 (2006) ; Mroczho et al., Clin. Chim. Acta 380: 208-212 (2007)). In one aspect of the invention, the level of CSF-1R ligand is at least 50% higher in cancer patient samples than in samples from the control population. The range of CSF-1 and / or IL-34 levels in the control population is determined by comparing the CSF-1 and / or IL-34 levels found in the patient sample or samples to the CSF- 1 < / RTI > and / or IL-34 levels.

In one aspect, the antibody of the invention is for use in treating cancer in which cancer cells are ligand-secreting. In yet another aspect, the antibody of the invention is for use in treating cancer in which the cancer cells are CSF-1-secreting. In yet another aspect, the antibody of the invention is for use in treating cancer in which the cancer cells are IL-34-secreting.

The invention also includes a kit for inhibiting tumor growth and / or angiogenesis, comprising a therapeutically effective amount of a human anti-CSF-1R antibody. The kit may comprise an antibody or a fragment thereof, and docetaxel, paclitaxel, Herceptin ^®, or the addition of agents to induce additional anticancer agent, an antineoplastic agent or therapy include, but not limited to doxorubicin. Alternatively or additionally, the kit may contain any suitable antagonist of another growth factor receptor involved in tumorigenesis or neovascularization as discussed below, for example. The kit of the present invention may further comprise a conjugate.

Thus, the receptor antibodies of the present invention can be used in vivo and in vitro for methods of detection, diagnosis, prophylaxis or treatment, which is well known in the art. Skilled artisans may make changes to the principles of the invention disclosed herein, and such variations are encompassed within the scope of the present invention.

It should be understood and appreciated by those of ordinary skill in the art that changes may be made in the principles of the invention disclosed herein and that such variations are included within the scope of the present invention.

Example

The following examples further illustrate the invention, but are not to be construed as limiting the scope of the invention in any manner, which should not be construed as limiting the broad scope of the invention in any way. Construction of vectors and plasmids, polypeptide coding for these vectors and plasmids, gene insertion, introduction of plasmids into host cells, and expression of the genes and gene products and their use in crystal determination are described in numerous publications , Cold Spring Harbor Laboratory Press (1989) and Coligan, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed. Current Protocols in Immunology, Wiley & Sons, Incorporated (2007).

Expression and purification of human anti-CSF-1R antibodies

For each antibody, a suitable heavy chain nucleotide sequence, such as SEQ ID NO: 13 for antibody 1, is engineered into a suitable expression plasmid such as pGSHC by a suitable method, such as PCR cloning, and the appropriate light chain nucleotide sequence, 1 was engineered into a suitable expression plasmid such as pGSLC. To establish a stable cell line, an appropriate host cell line such as NSO or CHO cells is co-transfected by electroporation with linear heavy and light chain plasmids and co-transfected into a suitable medium, for example, dialyzed fetal bovine serum and glutamine synthetase supplements Gt; Dulbecco's < / RTI > Modified Eagle Medium. Clones were screened for antibody expression by enzyme-linked immunosorbent assay (ELISA) and the best producing strains were selected for culture in spinner flasks. Antibodies were purified by suitable methods, such as protein-A affinity chromatography.

Table 1 provides the amino acid sequences and sequence numbers of the various CDRs of antibody 1 of the invention. All CDR sequences are determined using Kabat's custom. Table 2 provides SEQ ID NOs of various sequences related to the present invention. Polynucleic acid sequences encoding amino acid sequences disclosed below are also included within the scope of the present invention.

Enzyme-linked immunosorbent assay (ELISA) binding assay

CSF-1R-Fc fusion protein (1 < RTI ID = 0.0 >Lt; / RTI > After washing the wells three times with PBS (phosphate buffered saline) (PBS / T) containing 0.2% Tween-20 ^® , the wells were incubated for 1 hour at room temperature (20-25 ° C) (RT) with 3% bovine serum albumin 0.0 > (BSA). ≪ / RTI > The BSA-PBS mixture was aspirated and the plates washed three times with PBS / T. A series of dilutions of antibody 1 or control IgG were made (starting at 3 μg / mL and diluted 3-fold) and 100 μL was added to the wells for 1 hour at RT. Wells were washed three times with PBS / T. After washing, the plates were incubated for 1 hour at RT with 100 μL of anti-human F (ab ') ₂ fragment-specific-HRP conjugate (Jackson ImmunoResearch) (1: 5000 dilution) in PBS. Plates were washed five times with PBS / T and then incubated for 15 minutes at RT with 100 μL of a 1: 1 preparation of TMB peroxidase substrate and peroxidase solution B (KPL). 100 μL of 0.1 MH ₂ SO ₄ was added to stop the color reaction. Data were collected using a microplate reader set at 450 nm. ED ₅₀ was calculated by analyzing absorbance data with GraphPad Prism software. The ED ₅₀ or half the maximum effective capacity is the capacity required to achieve 50% maximal binding.

The ED ₅₀ of antibody 1 against human CSF-1R is 8.7 x ^{10 -11} M (recorded at 0.09 nM). Antibody 1 shows strong binding to CSF-1R.

Enzyme-Linked Immunosorbent Assay (ELISA) Blockade Assay

ELISA to show that antibodies block CSF-1 / CSF-1R interactions

96-well microtiter plates were coated overnight at 4 ° C with (0.5 μg / mL × 100 μL) CSF-1 (RAND Systems). Wells were washed three times with PBS / T and then blocked with 100 μL 3% BSA / PBS for 1 hour at RT. And then washed three times with PBS / T. Soluble human rh-CSF-1R-Fc fusion protein (R & D Systems) was diluted in PBS to a final concentration of 0.250 μg / mL. At the same time, antibody 1 was diluted in PBS to a final concentration of 200 nM. Antibody 1 was serially diluted in the 1: 3 increments from the initial 200 nM down to 3x10 ^-12 M. 100 μL of rh-CSF-1R-Fc was added to each dilution of 100 μL of antibody 1 at RT for 30 minutes. 100 [mu] L of antibody 1: CSF-1R-Fc mixture was incubated in the CSF-1-coated well for 1 hour at RT. After 1 hour of incubation at RT, the cells were washed three times with PBS / T and a 1: 5000 dilution of anti-human IgG-Fc-HRP conjugated antibody was added to the plate for 1 hour at RT. Plates were washed five times with PBS / T and then incubated with 100 [mu] L of a 1: 1 preparation of TMB peroxidase substrate and peroxidase solution B (KPL) for 15 min at RT. 100 μL of 0.1 MH ₂ SO ₄ was added to stop the color reaction _. Data were collected using a microplate reader set at 450 nm. The IC ₅₀ was calculated by analyzing the absorbance data with GraphPad prism software. The IC ₅₀ or half maximal inhibitory concentration is the concentration of antibody that causes 50% inhibition of ligand binding to the receptor.

The IC ₅₀ of antibody 1 binding to human CSF-IR is 8.1 x ^{10 -10} M (recorded at 0.81 nM). Antibody 1 inhibits CSF-1 binding to CSF-1R and prevents CSF-1R activation by CSF-1 ligand.

ELISA to show that antibodies block IL-34 / CSF-1R interactions

96-well microtiter plates were coated overnight at 4 [deg.] C with IL-34 (0.5 μg / mL x 100 μL). Wells were washed three times with PBS / T and then blocked with 100 μL 3% BSA / PBS for 1 hour at RT. And then washed three times with PBS / T. Soluble human rh-CSF-1R-Fc fusion protein (R & D Systems) was diluted in PBS to a final concentration of 0.250 μg / mL. At the same time, antibody 1 was diluted in PBS to a final concentration of 200 nM. Antibody 1 was serially diluted in the 1: 3 increments from the initial 200 nM down to 3x10 ^-12 M. 100 μL of rh-CSF-1R-Fc was added to each dilution of 100 μL of antibody 1 at RT for 30 minutes. 100 [mu] L of the antibody 1-CSF-1R-Fc mixture was incubated in the IL-34-coated well for 1 hour at RT. After 1 hour of incubation at RT, the cells were washed three times with PBS / T and a 1: 5000 dilution of anti-human IgG-Fc-HRP conjugated antibody was added to the plate for 1 hour at RT. Plates were washed five times with PBS / T and then incubated with 100 [mu] L of a 1: 1 preparation of TMB peroxidase substrate and peroxidase solution B (KPL) for 15 min at RT. 100 μL of 0.1 MH ₂ SO ₄ was added to stop the color reaction _. Data were collected using a microplate reader set at 450 nm. The IC ₅₀ was calculated by analyzing the absorbance data with GraphPad prism software. The IC ₅₀ or half maximal inhibitory concentration is the concentration of antibody that causes 50% inhibition of ligand binding to the receptor.

The IC ₅₀ of antibody 1 binding to human CSF-1R is 7.0 x ^{10 -10} M (recorded at 0.71 nM). Antibody 1 inhibits IL-34 binding to CSF-1R and prevents CSF-1R activation by IL-34 ligand.

Surface Plasmon Resonance / Biacore (Biacore) coupled dynamics analysis by analysis ^®

Binding kinetics of antibody 1 for CSF-1R-Fc was measured on a BIAcore ^® 2000 SPR biosensor (Biosensor) (GE Healthcare (GE Healthcare)) at 25 ℃. The soluble CSF-1R-Fc fusion protein (concentration 10 μg / mL and pH 5) (range 395-1200 reaction units) was immobilized on a CM5 chip using a standard amine coupling protocol. HBS-EP (0.01 M HEPES (pH 7.4), 0.15 mM NaCl, and 3 mM EDTA, 0.005% v / v surfactant P20) were used as running buffers during binding affinity measurements. Interaction analysis was performed by injecting antibodies in solution at a concentration ranging from 1.5-100 nM on the prepared surface of the CM5 sensor chip. Antibodies were injected for binding over 3 minutes and dissociated for 15 minutes. Regeneration of the immobilized protein was carried out by injection of 10 [mu] L / min of 20 mM HCL. Using the BIA evaluation version 4.1 software, K _a (k _on ) and K _d (k _off ) of the complex formation were determined by simultaneous global fitting to a 1: 1 Langmuir model of the data. The equilibrium association constant (K _A ) was calculated from the ratio of 1 / K _D (measured in molar (1 / M)). The equilibrium dissociation constant (K _D ) was calculated from the ratio of the rate constant K _d / K _a (measured in moles).

The average K _a, K _d and K _D values for multiple BIAcore ^® analysis for the first antibody has a human CSF-1R is summarized in Table 3.

Antibody 1 shows high binding affinity to CSF-1R.

The phosphorylation of CSF-IR detected by Western blot

NIH3T3 cells stably transfected with CSF-1R cells were cultured in 12-well plates at a density of ⁵ x 10 cells / well in a 1 mL well of Dulbecco's medium supplemented with 10% fetal bovine serum (FBS) and 1 mg / And seeded in modified Eagle's medium (DMEM) for 5 hours. The monolayer was washed twice in PBS and incubated overnight with 1% FBS in 0.9 mL / well DMEM. A series of dilutions of antibody 1 in DMEM (starting at 1 μg / mL and 3-fold dilution) were added and 100 μL was added to the wells for 2 hours at 37 ° C. Cells were stimulated with 100 ng / mL CSF-1 or IL-34 ligand for 10 minutes, then placed on ice and washed with ice-cold PBS. Cells were resuspended in 100 μL ice-cold 50 mM Hepes (pH 7.5 150 mM NaCl, 0.5% Triton X-100, 1 mM Na ₃ VO ₄ , 10 mM NaPPi, 50 mM NaF) and purification of the protease inhibitor (Roche) for 10 minutes. The lysed cells were purified by centrifugation at < RTI ID = 0.0 > 4 C. < / RTI > 20 [mu] L of the lysate was loaded onto the denaturing electrophoresis gel and blotted onto the nitrocellulose membrane. Tyrosine-phosphorylated CSF-1R on the blot was detected by the use of 1 μg / ml anti-phospho-CSF-1R antibody (Cell Signaling). CSF-1R signaling was determined by probing the blot with anti-phospho-MAPK (1: 500 dilution) or anti-phospho-Akt (1: 1000) (cell signaling). The probes were blotted with anti-CSF-1R antibody (1 μg / mL; NAND Systems) or anti-actin antibody (1: 2000; Sigma) to ensure the same loading of the lanes. After incubation for 1 hour at RT with shaking all primary antibodies, the corresponding secondary antibody conjugated to HRP was also incubated at RT for 1 hour with shaking. The band was visualized as a chemiluminescent reagent (geothermal).

NIH-3T3 cells stably transfected with CSF-1R showed rapid phosphorylation of CSF-1R when stimulated with CSF-1 or IL-34. The presence of antibody 1 also inhibited CSF-IR phosphorylation even when the level of antibody was 1 nM, indicating that binding of antibody 1 to CSF-1R prevents receptor activation by CSF-1 or IL-34 . Inhibition of CSF-IR phosphorylation also led to a reduction in the phosphorylation of signaling molecules used by the CSF-1 or IL-34 pathway. Both Akt and MAPK downstream of CSF1-R reduced phosphorylation when cells were incubated with 100 nM to 1 nM of antibody 1. Thus, antibody 1 prevents activation of the kinase cascade following CSF-IR activation and CSF-IR stimulation. There was no difference in protein levels of actin and CSF-1R between lanes, which is evidenced by the same chemiluminescent signal in each lane when blots were probed with antibodies to these molecules. Thus, the inhibition of CSF-IR phosphorylation is not due to technical difficulties in which protein samples are loaded heterogeneously, but actually shows reduced signal transduction.

Internalization and disassembly test

Antibody 1 induces internalization of the CSF-1R receptor

NIH-3T3-CSF-1R cells were plated in 8-well chamber slides (Nunc) and incubated at 37 占 until the cells covered 50% of the surface area of the wells. The experiment was started in a water: ice slurry mixture after the slides were cooled to 4 ° C. Cells were incubated with 5 μg / mL antibody 1 and immediately transferred to 37 ° C. for 15 minutes to 4 hours to allow for internalization. Separate sets of slides were incubated with 5 μg / mL antibody 1 as a control for 15 minutes to 4 hours in a water: ice slurry mixture. The 4 ° C incubation prevents internalization of the CSF-1 receptor, and thus any signal that appears within the cell is an artifact. After incubation, the cells were washed twice with cold PBS, and then the cells were fixed with 1% paraformaldehyde in ice-cold for 15 minutes. After 3 washes with cold PBS, the cells were permeabilized for 5 minutes in PBS containing 0.5% saponin and 1% BSA, followed by washing again in PBS containing 1% BSA. Antibody 1 was fluorescently labeled with goat Cy3 anti-human IgG (1: 5000 dilution) for 1 hour. Finally, after three washes with PBS, mounting was performed on a GelMount (Biomeda) and the slides were covered with a cover glass. Fluorescently labeled antibody 1 can be visualized under a microscope to determine the cell location under the various conditions described above.

At the microscopic examination, antibody 1 was observed at the periphery of the cells at 4 째 C before the start of the experiment. Cells were switched to 37 ° C to permit rapid internalization of the antibody 1 / CSF-1R complex, which is generally observed within 15 minutes. After incubation for 1 or 2 hours with antibody 1, all antibody / CSF-1R complexes were localized around the nucleus with very little visualization on the plasma membrane, indicating that binding of antibody 1 to CSF-1R resulted in rapid internalization of the receptor, ≪ / RTI > Cells maintained at 4 [deg.] C for up to 2 hours showed only peripheral staining of cells without antibody 1 internalization. Thus, the internalization of antibody / CSF-1R is an ATP-dependent process that takes place within 15 minutes after the addition of antibody to the cells.

Antibody 1 induces degradation of the CSF-1R receptor

NIH-3T3-CSF-1R cells were seeded in 12-well plates at ⁵ x 105 cells / well in 1 mL DMEM medium containing 10% FBS and 1 mg / mL geneticin. After incubating the cells for 5 hours at 37 ° C, 100 ng / mL CSF-1 or 15 μg / mL antibody 1 was added to the plate. CSF-1 is known to induce internalization and degradation of CSF-1R, thereby serving as a positive control in the experiment. CSF-1 was allowed to sit on the cells for 1 and 5 hours before cells were harvested. In the other wells, cells were harvested after incubation with antibody 1 for 24 and 48 hours. The medium was aspirated, the cells were lysed and the clarified lysate was run on a gel (as described above). The delivered protein was probed with anti-CSF-1R antibody to determine the amount of receptor for each condition. The same blot was probed with anti-actin antibody to normalize protein levels. CSF-1R and actin levels were quantified using a Multi-Gauge program to determine band density on Western blot. The relative total CSF-1R protein level is expressed as the total CSF-1R band density divided by the corresponding actin band density.

CSF-1 incubation with NIH-3T3-CSF-1R cells leads to the degradation of CSF-1R within 1 hour of treatment and CSF-1R level is halved in 5 hours. When incubated with antibody 1, degradation of CSF-1R was not apparent, but antibody 1 reduced the level of CSF-1R by a factor of four. After 48 hours, the degradation level remained the same, indicating that the decomposition rate remained constant. Thus, antibody-1 binding to CSF-1R leads to degradation of the receptor in the cell.

Antibody-dependent cell-mediated cytotoxicity (ADCC)

Antibody 1 inhibits CSF-1R by inhibiting CSF-1 binding to CSF-1R and inducing the internalization and degradation of CSF-1R, as well as by causing an ADCC reaction.

NKM-1 human leukemia cells were seeded in 25 μL RPMI medium containing 10% Ultra-Low IgG FBS and 3 ng / mL human IL-2 in 96 well plates at 2 × 10 ⁵ cells / mL . A 1: 3 serial dilution of 1 μg / mL antibody 1 in IgG1 or IgG2 backbones was made and 25 μL / well was added. After 15 minutes of incubation, 25 μL of 3 × 10 ⁶ cells / mL human NK cells (Lonza) were seeded and incubated at 37 ° C. for an additional 4 hours. 10 μL of lysis buffer was added from the aCella-TOX kit (Cell Technology) and allowed to RT for 15 minutes. 125 [mu] L of low IgG FBS was added and the plate was centrifuged at 750 xg for 1 minute. In Optiplate (Perkin Elmer), 50 [mu] L enzyme assay diluent was added from the Acella-Toxok kit and 50 [mu] L of cell supernatant was carefully transferred to the Optiplate. An enzyme assay reagent and a detection reagent were prepared according to the kit manual, and 100 μL of the enzyme assay reagent was added to the Optiplate, and immediately 50 μL of the detection reagent was added. After 20 minutes the reagents were added and the plates read in the luminescence meter.

ADCC activity was increased in an antibody 1 dose-dependent manner to 9% of killed NKM-1 cells when antibody 1 concentration reached 1 μg / mL. In contrast, ADCC activity did not change with increasing amounts of antibody 1 when antibody 1 was cloned into the IgG2 backbone. Thus, antibody 1, IgG1 molecule induces ADCC of binding cells.

Epitope mapping

Previous publications have determined that CSF-1 binds to the first three Ig domains of CSF-1R. Wang et al., Mol. Cell. Biol. 13: 5348 (1993)]. Antibody 1 binds to CSF-1R and inhibits CSF-1 binding to human CSF-1R, which should bind to or near the first three Ig domains of CSF-1R. To determine if epitope antibody 1 binds on the CSF-1R molecule, the first three Ig domains of the mouse and human were exchanged (see Table 4). Antibody 1 did not recognize the murine CSF-1R, and if the murine Ig domain was inserted into the critical binding region of human CSF-1R, binding of the antibody would not occur.

Modifications of the first three Ig domains were inserted into the human CSF-1R backbone containing the two most C-terminal Ig domains of the extracellular domain. These constructs were fused to the Fc portion of the IgG molecule for ease of protein production and stability of the molecule.

Thus, chimeric 1 contains mouse Ig domain 1, human Ig domain 2 and human Ig domain 3 (m, h, h), human Ig domains 4 and 5 (fused to an Fc tag). M, m, m (chimeric 3), h, m, h (chimeric 4), h, m, m (chimeric 5) h, h, m (chimeric 6) and m, h, m (chimeric 7) fused to the Fc tag following the last two Ig domains of human CSF-1R.

Chimeric antibody binding 1 for CSF-1R protein was determined by ELISA binding and Biacore ^® as described above. Briefly, plates were coated overnight with 100 μL of 200 ng / mL human, mouse or chimeric 1-7 protein. After washing and blocking of the plate, antibody 1 was repeatedly added at a concentration of 100 nM. HRP conjugated anti-human Fab secondary antibody was added at a concentration of 1: 10,000 to detect antibody 1 binding to CSF-1R molecules.

As expected, antibody 1 bound to human CSF-1R but not to mouse CSF-1R in ELISA binding assays. Antibody 1 bound weakly to chimeras containing the first or second human Ig domain, but not to those containing the first or second mouse Ig domain. Antibody 1 binds strongly to chimeras containing both the first and second Ig domains of human CSF-1R. All other constructs did not bind to antibody 1. Thus, antibody 1 binds to Ig domains 1 and 2, but both domains are required for strong antibody binding to human CSF-1R as assessed in binding assays. Biacore ^® data summarizes ELISA data. Any chimeric construct that does not contain the human second Ig domain did not bind to antibody 1 even when the antibody level was 1 [mu] M, indicating that the second Ig domain is required for antibody 1 binding. Antibody 1 binding was further enhanced when the first Ig domain was also of human origin. Thus, antibody 1 binds primarily to the second Ig domain of CSF-IR, but the first Ig domain has some beneficial contact.

Differentiation and proliferation assay

Macrophage differentiation by CSF-1

Monocytes (theorists) 4x10 ⁵ Were seeded in 900 μL of Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS and 100 ng / mL hCSF-1 in each chamber of an 8-chamber slide at a density of cells / mL. A series of dilutions of antibody 1 in RPMI were made (starting at 1 μg / mL and 3-fold dilution), and 100 μL was added to the wells. The cells were incubated at 37 [deg.] C with varying media every 3 days until monocytes were clearly attached to the plate and appeared to elongate (characteristic of macrophage differentiation).

Monocytes treated with antibody 1 at a concentration of 2 nM or higher in the presence of CSF-1 failed to differentiate into macrophages and retained their monocyte morphology. CSF-1 induction by macrophage differentiation of monocytes can be inhibited by antibody 1 to an IC ₅₀ of 0.25 nM. In addition, many died during the treatment and could not survive without continued stimulation with CSF-1.

Macrophage differentiation by IL-34

Monocytes (theorists) 4x10 ⁵ Were seeded in 900 μL of Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS and 100 ng / mL hIL-34 in each chamber of an 8-chamber slide at the density of cells / mL. A series of dilutions of antibody 1 in RPMI were made (starting at 1 μg / mL and 3-fold dilution), and 100 μL was added to the wells. The cells were incubated at 37 [deg.] C with varying media every 3 days until monocytes were clearly attached to the plate and appeared to elongate (characteristic of macrophage differentiation).

Monocytes treated with antibody 1 at a concentration of 2 nM or higher in the presence of IL-34 failed to differentiate into macrophages and retained their monocyte morphology. IL-34 induction into macrophage differentiation of monocytes can be inhibited by antibody 1 with an IC ₅₀ of 0.3 nM. In addition, many died during the treatment and could not survive without continued stimulation with CSF-1.

Monocyte proliferation by CSF-1

Monocytes were seeded in 96-well plates in the presence of RPMI 1640 medium containing 10% FBS and 100 ng / mL CSF-1 at a density of 3000 cells / well. The next day, the medium was changed and antibody 1 (1: 3 dilution) diluted successively from 20 nM to 0.01 nM was added. After 3 days, the medium was replaced with fresh medium containing 10% FBS, 100 ng / mL CSF-1 and antibody, and the cells were grown for an additional 5 days. Upon addition of 100 [mu] L of CellTiter Glo luminescent buffer and substrate (Promega), cells were shaken for 10 minutes and luminescence read as an indicator of viability.

The IC ₅₀ of antibody 1, which inhibits 50% of mononuclear growth, is 1.4 x ^{10 -10} M (recorded at 0.1 nM). The IC ₅₀ for CSF-1 induction of monocyte growth in the presence of antibody 1 indicates that antibody 1 inhibits monocyte proliferation in the culture.

Monocyte proliferation by IL-34

Monocytes were seeded in 96-well plates in the presence of RPMI 1640 medium containing 10% FBS and 100 ng / mL IL-34 at a density of 3000 cells / well. The next day, the medium was changed and antibody 1 (1: 3 dilution) diluted successively from 20 nM to 0.01 nM was added. After 3 days, the medium was replaced with fresh medium containing 10% FBS, 100 ng / mL IL-34 and antibody and the cells were grown for an additional 5 days. Upon addition of 100 [mu] L of the cell lysozyme luminescent buffer and substrate (Promega), the cells were shaken for 10 minutes and luminescence read as an indicator of viability.

The IC ₅₀ of antibody 1, which inhibits 50% of mononuclear growth, is 0.5 nM. The IC ₅₀ for IL-34 induction of monocyte growth in the presence of antibody 1 indicates that antibody 1 inhibits monocyte proliferation in the culture.

Proliferation Assay for Tumor Cell Lines Containing CSF-1

NKM-1 leukemia cells were seeded overnight in 100 μL RPMI 1640 medium containing 1% FBS in 96-well plates at 1 × 10 ⁴ cells / mL. 20 ng / mL of CSF-1 was added to the cells in RPMI 1640 medium containing 1% FBS and antibody 1 (1: 3 dilution) diluted continuously from 20 nM to 0.01 nM. Lt; RTI ID = 0.0 > 37 C < / RTI > for an additional 3 days. Upon addition of 100 [mu] l of the cell lysozyme luminescent buffer and substrate (Promega), luminescence (as an indicator of viability) was read after shaking the cells for 10 min.

The IC ₅₀ for NKM-1 growth in the presence of antibody 1 is 7.6 x ^{10 -11} M (recorded at 0.07 nM). Indicating that antibody 1 inhibits the proliferation of NKM-1 cells in culture.

In vivo tumor model in which CSF-1R is expressed on the surface of the tumor

Survival of NKM-1 human leukemia-bearing mice treated with antibody 1

Nu / nu mice were irradiated with nearly lethal dose at 200 rad / mouse for 24 hours followed by intravenous injection of 2.5x10 ⁶ NKM-1 leukemia cells. After an additional 24 hours, mice were randomly divided into four groups receiving 60 mg / kg human IgG, 60 mg / kg, 20 mg / kg or 5 mg / kg antibody 1 twice a week. Survival of the mice was monitored daily. Log rank The P value was determined by the Mantel Cox test.

The tumor volume was calculated by the formula: volume = [(Pi / 6) lxw ² ], where Pi is 3.14, w is the width and l is the length. Percent of control or% T / C = 100 * (treatment volume) / (control volume). If the p-value is less than 0.05, statistical significance is assumed.

Antibody 1 increases the survival of mice bearing NKM-1 leukemia cells as shown in Table 5.

In vivo tumor models in which CSF-1R is expressed on the surface of tumor-associated macrophages

Anti-CSF-1R antibody 1 is a fully human antibody that recognizes the human form of CSF-1R but does not recognize the murine form of the receptor. Thus, anti-mouse antibodies are required for the implementation of proof of concept in in vivo studies in which CSF-1R is expressed on macrophages. These studies address the role of mouse macrophages in the growth of human tumors in a mouse xenograft model. Anti-mouse antibodies affect mouse macrophages but not tumors, which allows the ability to measure the effect of substrate-macrophages on tumor growth. The following experiment was performed with antibody 2, rat-anti-mouse CSF-1R antibody.

The efficacy of anti-mouse CSF-1R mAb in the AN3CA xenograft model of human endometrial carcinoma

Nu / nu mice (female, 7-8 weeks old) were injected subcutaneously with 5x10 ⁶ AN3CA cells / mouse. When tumors reached approximately 200 mm ³ , mice were randomly divided into 12 mice / treatment groups.

Antibody 2 and rat IgG were prepared at a concentration of 6 mg / mL in saline and subcutaneously administered at 40 mg / kg three times per week. Tumor volume was recorded at day 15 and% T / C was calculated. The tumor volume was analyzed using RM ANOVA. A T / C% of 66% was calculated for the treated group versus the rat IgG control for antibody 2. These results show significant tumor suppression in animals treated with antibody 2 (p = 0.045).

Efficacy of anti-mouse CSF-1R mAb in HCC1954 xenograft model of human breast carcinoma

Nu / nu mice (female, 7-8 weeks; Charles River Laboratories) mice were injected subcutaneously with 1 x 10 ⁷ HCC1954 cells / mouse. When the tumor size reached 300 mm ³ , mice were randomly assigned by tumor size, with 12 animals assigned to each treatment group. Rat IgG and antibody 2 were prepared at a concentration of 6 mg / mL in saline and subcutaneously administered three times per week. Rat IgG was administered to the animals at 40 mg / kg, and antibody 2 was administered to the animals at 40 mg / kg or 10 mg / kg at each injection. Tumor measurements were recorded twice a week and T / C% was calculated 37 days after the first injection. The tumor volume was analyzed using RM ANOVA.

As shown in Table 6, in the treatment group treated with 10 mg / kg as the ratio of the relative tumor volume to the saline control on the 37th day, treatment group treated with 77% T / C% and 40 mg / kg 56% T / C% was calculated. The results show significant tumor suppression in animals treated with 40 mg / kg antibody 2 (p = 0.0014).

Antitumor effect of anti-mouse CSF-1R mAb in DU145 human prostate xenograft model

Nu / nu mice (male, 7-8 weeks old) were injected subcutaneously with 15x10 ⁶ DU145 cells / mouse. When tumors reached approximately 250 mm ³ , mice were randomly divided into 12 mice / treatment groups.

Antibody 2 and rat IgG were prepared at a concentration of 6 mg / mL in saline and subcutaneously administered at 40 and 10 mg / kg three times per week. Tumor volume was recorded on day 21 and% T / C was calculated for 10 mg / kg and 40 mg / kg. The tumor volume was analyzed using RM ANOVA.

As shown in Table 6, T / C% of 50 and 43 in the treatment group 10 mg / kg and 40 mg / kg versus the rat IgG control group, respectively, were calculated. These results show significant tumor suppression in animals treated with antibody 2 (p = <0.0001 for both concentrations).

Reduction of macrophage infiltration in HCC1954 breast and DU145 prostate tumors treated with anti-mouse CSF-1R mAb

Tumors were removed from the animals in the HCC1954 (breast) and DU145 (prostate) animal studies described above. Tumors were cut along the longest axis and placed at 4 ° C overnight shaking in 10% formalin. Twenty-four hours later, after two washes in PBS for 20 minutes, a higher concentration of ethanol solution was gradually added until the tumors were in 100% ethanol. After several incubations in 100% xylene, ethanol was replaced by xylene and the tumor was embedded in paraffin. The paraffin block was cut at 4 μm and placed on a glass slide. The tissues were deparaffinized and rehydrated by xylene followed by gradual incubation in increasing concentrations of ethanol in water. Tumor slides were heated in microwave for 3 min in a target restoration solution (Dako) and endogenous peroxidase was blocked with 3% H ₂ O ₂ for 10 min at RT. The non-specific protein binding was blocked with protein block (Dako) for 10 min followed by addition of biotin-conjugated rat anti-mouse macrophage-specific antibody, F4 / 80 (2 μg / mL; Serotec) Lt; RTI ID = 0.0 &gt; 4 C &lt; / RTI &gt; overnight. HRP-streptavidin (1: 1000 dilution; Jackson Immunoresearch) for 45 min at RT and washed three times. The color was developed in DAB (Dako) according to the kit instructions, and the reaction was stopped by washing twice in water. Briefly stained with Mayer hematoxylin (10 min; Dako), then washed with water, immersed in acid alcohol, washed twice with water, and blueed with ammonia water. Dehydrated, cleaned and covered with a cover slip using a permanent mounting medium. Five images from three animals were analyzed for each treatment group. Using ImagePro software, the number / area of macrophages was determined for each treatment group.

As shown in Table 7, macrophage counts decreased in both tumor models, particularly along the periphery. Thus, treatment with antibody 2 leads to a decrease in macrophage infiltration of the tumor, indicating that the anti-mouse CSF-1R antibody is responsible for the depletion of the macrophage population in these tumors.

Significance of macrophage and CSF-1 levels on tumor progression

In addition to breast and prostate gland, treatment of multiple cancers as well as inhibition of tumor growth may be beneficially effected by the administration of antibody 1. Some tumor types, such as renal cancer, express CSF-1R on its cell surface and growth can be directly inhibited by antibody 1 treatment (Soares, et al., Modern Pathol. 22: 744-752 (2009) ). Other tumor types with large tumor-associated macrophage populations, such as Hodgkin's lymphoma and multiple myeloma, may benefit from treatment with antibody 1, where antibody 1 can remove macrophages that induce tumor growth (Steidl Zheng, et al., Blood 114: 3625-3628 (2009)). In addition, tumors that secrete CSF-1, such as colorectal cancer, are susceptible to anti-CSF-1 or CSF-1R treatment (Aharinejad, et al., Cancer Res. 62: 5317-5324 (2002) , And would be suitable for antibody 1 treatment. In addition, ovarian cancer, hepatocellular carcinoma and renal cell carcinoma associated with poor prognosis of high CSF-1 expression will all be candidates for antibody 1 treatment (Toy, et al., Gyn. Oncol. 80: 194- Zhu, et al., J. Clin. Oncol. 26: 2707-2716 (2008); Gerharz, et al. , Urol, 58: 821-827 (2001)).

Non-CSF-1R expressing tumors with tumor-associated macrophages are growth inhibited by anti-CSF-1R antibodies only when CSF-1 secreted

Tumor cell lines HCC1954 (breast), DU145 (prostate) and Pc3 (prostate) were seeded in growth media containing 1% FBS for 48 hours at ⁵ x 10 cells / mL. The medium was harvested, purified and analyzed using the R & D Quantikine human M-CSF assay kit (R & D Systems). CSF-1 levels were determined as pg / mL at 0 and 48 hours.

At time 0, there was no discernable CSF-1 in the medium as expected. However, within 48 hours, HCC 1954 cells secreted 3613 pg / mL of CSF-1 while DU145 cells produced 4019 pg / mL. In contrast, Pc3 cell culture medium contained only 39 pg / mL of CSF-1.

Prostate cell line Pc3 was grown in mice to confirm the lack of CSF-1 expression correlated with resistance to anti-CSF-1R treatment

5x10 ⁶ Pc3 prostate cells were subcutaneously injected into nude mice (male, 7-8 weeks old), and the tumors reached 300 mm &lt; ^{3 &} gt ;, then divided into treatment groups of 12 mice each. Animals were administered 40 mg / kg of rat IgG, 40 mg / kg of antibody 2 or 10 mg / kg of antibody 2 three times a week until the control tumors reached 2000 mm ³ . Tumor volume was measured and at day 18, T / C% for each treatment group was calculated as the ratio of relative tumor volume to rat IgG control. The tumor volume was analyzed using RM ANOVA.

There was no difference in growth between the rats IgG treated control vs. antibody 2 treated groups, indicating that elevated levels of CSF-1 may be biomarkers for treatment with anti-CSF-1R antibodies.

CSF-1 secretion correlates with the susceptibility of anti-CSF-1R treatment when CSF-1R is expressed on the surface of tumor-associated macrophages

Cells (described in Tables 9 and 10 below) were subcutaneously injected into nude mice and subdivided into treatment groups of 12 mice each after allowing tumors to reach 300 mm ³ . Animals were dosed three times per week according to the treatment regimen described in Tables 9 and 10 below until the control tumors reached 2000 mm &lt; ³ &gt;. Tumor volume was measured and T / C% for each treatment group was calculated as the ratio of relative tumor volume to rat IgG control. The tumor volume was analyzed using RM ANOVA.

CSF-1 secreted tumors (Table 9) all respond to anti-CSF-1R treatment with antibody 2, whereas tumors that do not secrete CSF-1 (Table 10) do not respond to anti- Do not respond. CSF-1 secretion is associated with the susceptibility of anti-CSF-1R treatment in a tumor model in which CSF-1R is expressed on the surface of tumor-associated macrophages. Thus elevated levels of CSF-1 function as potential susceptibility indicators or biomarkers when CSF-IR is expressed on the surface of tumor-associated macrophages.

CSF-IR can also be expressed on the surface of tumor cells. CSF-1 secretion is not related to the susceptibility of anti-CSF-1R treatment to CSF-1R tumor cell surface expression.

IL-34 levels in tumor and patient serum samples

In addition to CSF-1, IL-34 also binds to CSF-1R, induces phosphorylation of the receptor, and activates downstream signaling molecules, leading to macrophage differentiation and survival. Both CSF-1 and IL-34 bind to the IgG domain in the extracellular domain of CSF-1R (Lin et al., Science, 320: 807-11 (2008)), 1 (as discussed in the respective references, IC ₅₀ = 0.81 nM and IC ₅₀ = 0.71 nM for inhibition of CSF-1 and IL-34 binding by antibody 1, respectively ). Treatment of NIH-3T3 cells stably transfected with CSF-1R with antibody 1 inhibits the phosphorylation of CSF-1R by IL-34 (as discussed in the literature). In addition, IL-34 induction by macrophage differentiation and macrophage proliferation of monocytes was inhibited by antibody 1 with an IC ₅₀ of 0.3 nM and 0.5 nM, respectively (as discussed in the literature), by CSF-1 It is comparable to the results shown. Thus, IL-34 activity and its inhibition by antibody 1 are similar to those exhibited by CSF-1.

IL-34 levels are elevated in many tumor cells, including breast, prostate, and endometrial tumor cell lines. In addition, subgroups of prostate and breast patients have elevated IL-34 protein levels in their serum. Thus, elevated levels of IL-34 also function as a susceptibility indicator or biomarker, since IL-34 functions approximately the same as CSF-1 and CSF-1 secretion is associated with the susceptibility of anti-CSF-1R treatment.

Breast Tumor Combination Study

Nu / nu mice (female, 7-8 weeks old) received 1 x 10 ⁷ HCC-1954 breast cells / mice were subcutaneously injected and allowed the tumor to reach 300 mm ³ before treatment. Mice were randomly grouped into 12 groups and treated as follows:

Tumor volume was measured and T / C% for each treatment group was calculated as the ratio of relative tumor volume to control. The tumor volume was analyzed using RM ANOVA.

In all the studies shown in Table 11, antibody 2, Herceptin ^®, doxorubicin and paclitaxel inhibited tumor growth as a single agent. However, combining antibody 2 with a tumor-targeting agent has an additive effect that indicates that the combination of an anti-CSF-1R antibody with a chemotherapeutic agent or other agonist that affects the tumor would be more potent than these reagents alone.

Prostate tumor combination study

Nu / nu mice (male, 7-8 weeks old) received 1.5x10 ⁷ DU145 prostate cells / mice were subcutaneously injected and allowed the tumor to reach 300 mm ³ before treatment. Mice were randomly grouped into 10 groups and treated as follows:

Tumor volume was measured and T / C% for each treatment group was calculated as the ratio of relative tumor volume to control. The tumor volume was analyzed using RM ANOVA.

Combining antibody 2 with docetaxel has an additive effect in prostate cancer indicating that the combination with chemotherapeutic agent will have a higher potency than the chemotherapeutic agent alone.

Additional sequences

                         SEQUENCE LISTING <110> Eli Lilly and Company   <120> ANTIBODIES AGAINST CSF-1R <130> X18903 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 1 Ser Tyr Gly Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 2 Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 3 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 3 Gly Asp Tyr Glu Val Asp Tyr Gly Met Asp Val 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 4 Arg Ala Ser Gln Gly Ile Ser Asn Ala Leu Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 5 Asp Ala Ser Ser Leu Glu Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 6 Gln Gln Phe Asn Ser Tyr Pro Trp Thr 1 5 <210> 7 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 7 Gln Asp Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Asp Tyr Glu Val Asp Tyr Gly Met Asp Val Trp Gly Gln             100 105 110 Gly Thr Thr Val Thr Val Ala Ser         115 120 <210> 8 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 8 Ala Ile Gln Leu Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Ala             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Trp                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 9 <211> 450 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 9 Gln Asp Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Asp Tyr Glu Val Asp Tyr Gly Met Asp Val Trp Gly Gln             100 105 110 Gly Thr Thr Val Thr Val Ala Ser Ala Ser Thr Lys Gly Pro Ser Val         115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala     130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val                 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro             180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys         195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp     210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile                 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu             260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His         275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg     290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu                 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr             340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu         355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp     370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp                 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His             420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro         435 440 445 Gly Lys     450 <210> 10 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 10 Ala Ile Gln Leu Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Ala             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Trp                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 11 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 11 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Asp Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln             20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe         35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Glu Gly Leu     50 55 60 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Gly Asp Tyr Glu Val Asp Tyr Gly Met Asp Val         115 120 125 Trp Gly Gln Gly Thr Thr Val Thr Val Ala Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val     210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu                 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr             260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val         275 280 285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val     290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu                 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala             340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro         355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln     370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr                 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu             420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser         435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser     450 455 460 Leu Ser Pro Gly Lys 465 <210> 12 <211> 233 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 12 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala             20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile         35 40 45 Ser Asn Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys     50 55 60 Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Ser Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser                 85 90 95 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Cyr Gln Gln Phe Asn Ser             100 105 110 Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr         115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu     130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly                 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr             180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His         195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val     210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 13 <211> 1407 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 13 atgggatggt catgtatcat cctttttctg gtagcaactg caactggagt acattcacag 60 gaccagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120 tgtgcagcgt ctggattcac cttcagtagc tatggcatgc actgggtccg ccaggctcca 180 ggcgaggggc tggagtgggt ggcagttata tggtatgatg gaagtaataa atactatgca 240 gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac actgtatctg 300 caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag aggtgactac 360 gaggtcgact acggaatgga cgtctggggc caagggacca cggtcaccgt cgcctcagct 420 agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagagag tgagcccaaa 720 tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 780 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840 gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtat 900 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 960 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccaag actggctgaa tggcaaggag 1020 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1080 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg 1140 accaagaacc aagtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260 gctccgacg gctccttctt cctctattcc aagctcaccg tggacaagag caggtggcag 1320 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380 aagagcctct ccctgtctcc gggcaaa 1407 <210> 14 <211> 838 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 14 atgggatggt catgtatcat cctttttcta gtagcaactg caactggagt acattcagcc 60 atccagttga cccagtctcc atcctccctg tctgcatctg taggagacag agtcaccatc 120 acttgccggg caagtcaggg cattagcaat gctttagcct ggtatcagca gaaaccaggg 180 aaagctccta agctcctgat ctatgatgcc tccagtttgg aaagtggggt cccatcaagg 240 ttcagcggca gtggatctgg gacagatttc actctcacca tcagcagcct gcagcctgaa 300 gattttgcaa cttattactg tcaacagttt aatagttacc cgtggacgtt cggccaaggg 360 accaaggtgg aaatcaaacg tgagttctag aggatccatc tgggataagc atgctgtttt 420 ctgtctgtcc ctaacatgcc ctgtgattat ccgcaaacaa cacacccaag ggcagaactt 480 tgttacttaa acaccatcct gtttgcttct ttcctcagga actgtggctg caccatctgt 540 cttcatcttc ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct 600 gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca 660 atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct 720 cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga 780 agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgt 838 <210> 15 <211> 972 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 15 Met Gly Pro Gly Val Leu Leu Leu Leu Leu Val Ala Thr Ala Trp His 1 5 10 15 Gly Gln Gly Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu Val Val             20 25 30 Lys Pro Gly Ala Thr Val Thr Leu Arg Cys Val Gly Asn Gly Ser Val         35 40 45 Glu Trp Asp Gly Pro Ala Ser Pro His Trp Thr Leu Tyr Ser Asp Gly     50 55 60 Ser Ser Ile Leu Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly 65 70 75 80 Thr Tyr Arg Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala                 85 90 95 Ile His Leu Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala             100 105 110 Gln Glu Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu         115 120 125 Leu Thr Asp Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg     130 135 140 Gly Arg Pro Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp His 145 150 155 160 Gly Phe Thr Ile His Arg Ala Lys Phe Ile Gln Ser Gln Asp Tyr Gln                 165 170 175 Cys Ser Ala Leu Met Gly Gly Arg Lys Val Met Ser Ile Ser Ile Arg             180 185 190 Leu Lys Val Gln Lys Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val         195 200 205 Pro Ala Glu Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys     210 215 220 Ser Ala Ser Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn 225 230 235 240 Asn Thr Lys Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg                 245 250 255 Tyr Gln Lys Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His             260 265 270 Ala Gly Asn Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys His Ser         275 280 285 Thr Ser Met Phe Phe Arg Val Val Glu Ser Ala Tyr Leu Asn Leu Ser     290 295 300 Ser Glu Gln Asn Leu Ile Gln Glu Val Thr Val Gly Glu Gly Leu Asn 305 310 315 320 Leu Lys Val Met Val Glu Ala Tyr Pro Gly Leu Gln Gly Phe Asn Trp                 325 330 335 Thr Tyr Leu Gly Pro Phe Ser Asp His Gln Pro Glu Pro Lys Leu Ala             340 345 350 Asn Ala Thr Thr Lys Asp Thr Tyr Arg His Thr Phe Thr Leu Ser Leu         355 360 365 Pro Arg Leu Lys Pro Ser Glu Ala Gly Arg Tyr Ser Phe Leu Ala Arg     370 375 380 Asn Pro Gly Gly Trp Arg Ala Leu Thr Phe Glu Leu Thr Leu Arg Tyr 385 390 395 400 Pro Pro Glu Val Ser Val Ile Trp Thr Phe Ile Asn Gly Ser Gly Thr                 405 410 415 Leu Leu Cys Ala Ala Ser Gly Tyr Pro Gln Pro Asn Val Thr Trp Leu             420 425 430 Gln Cys Ser Gly His Thr Asp Arg Cys Asp Glu Ala Gln Val Leu Gln         435 440 445 Val Trp Asp Asp Pro Tyr Pro Glu Val Leu Ser Gln Glu Pro Phe His     450 455 460 Lys Val Thr Val Gln Ser Leu Leu Thr Val Glu Thr Leu Glu His Asn 465 470 475 480 Gln Thr Tyr Glu Cys Arg Ala His Asn Ser Val Gly Ser Gly Ser Trp                 485 490 495 Ala Phe Ile Pro Ile Ser Ala Gly Ala His Thr His Pro Pro Asp Glu             500 505 510 Phe Leu Phe Thr Pro Val Val Ala Cys Met Ser Ile Met Ala Leu         515 520 525 Leu Leu Leu Leu Leu Leu Leu Leu Leu Tyr Lys Tyr Lys Gln Lys Pro     530 535 540 Lys Tyr Gln Val Arg Trp Lys Ile Ile Glu Ser Tyr Glu Gly Asn Ser 545 550 555 560 Tyr Thr Phe Ile Asp Pro Thr Gln Leu Pro Tyr Asn Glu Lys Trp Glu                 565 570 575 Phe Pro Arg Asn Asn Leu Gln Phe Gly Lys Thr Leu Gly Ala Gly Ala             580 585 590 Phe Gly Lys Val Val Glu Ala Thr Ala Phe Gly Leu Gly Lys Glu Asp         595 600 605 Ala Val Leu Lys Val Ala Val Lys Met Leu Lys Ser Thr Ala His Ala     610 615 620 Asp Glu Lys Glu Ala Leu Met Ser Glu Leu Lys Ile Met Ser His Leu 625 630 635 640 Gly Gln His Glu Asn Ile Val Asn Leu Leu Gly Ala Cys Thr His Gly                 645 650 655 Gly Pro Val Leu Val Ile Thr Glu Tyr Cys Cys Tyr Gly Asp Leu Leu             660 665 670 Asn Phe Leu Arg Arg Lys Ala Glu Ala Met Leu Gly Pro Ser Leu Ser         675 680 685 Pro Gly Gln Asp Pro Glu Gly Gly Val Asp Tyr Lys Asn Ile His Leu     690 695 700 Glu Lys Lys Tyr Val Arg Arg Asp Ser Gly Phe Ser Ser Gln Gly Val 705 710 715 720 Asp Thr Tyr Val Glu Met Arg Pro Val Ser Thr Ser Ser Asn Asp Ser                 725 730 735 Phe Ser Glu Gln Asp Leu Asp Lys Glu Asp Gly Arg Pro Leu Glu Leu             740 745 750 Arg Asp Leu Leu His Phe Ser Ser Gln Val Ala Gln Gly Met Ala Phe         755 760 765 Leu Ala Ser Lys Asn Cys Ile His Arg Asp Val Ala Ala Arg Asn Val     770 775 780 Leu Leu Thr Asn Gly His Val Ala Lys Ile Gly Asp Phe Gly Leu Ala 785 790 795 800 Arg Asp Ile Met Asn Asp Ser Asn Tyr Ile Val Lys Gly Asn Ala Arg                 805 810 815 Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Cys Val Tyr             820 825 830 Thr Val Gln Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile         835 840 845 Phe Ser Leu Gly Leu Asn Pro Tyr Pro Gly Ile Leu Val Asn Ser Lys     850 855 860 Phe Tyr Lys Leu Val Lys Asp Gly Tyr Gln Met Ala Gln Pro Ala Phe 865 870 875 880 Ala Pro Lys Asn Ile Tyr Ser Ile Met Gln Ala Cys Trp Ala Leu Glu                 885 890 895 Pro Thr His Arg Pro Thr Phe Gln Gln Ile Cys Ser Phe Leu Gln Glu             900 905 910 Gln Ala Gln Glu Asp Arg Arg Glu Arg Asp Tyr Thr Asn Leu Pro Ser         915 920 925 Ser Ser Arg Ser Gly Gly Ser Gly Ser Ser Ser Ser Glu Leu Glu Glu     930 935 940 Glu Ser Ser Glu His Leu Thr Cys Cys Glu Gln Gly Asp Ile Ala 945 950 955 960 Gln Pro Leu Leu Gln Pro Asn Asn Tyr Gln Phe Cys                 965 970 <210> 16 <211> 972 <212> PRT <213> Homo sapiens <400> 16 Met Gly Pro Gly Val Leu Leu Leu Leu Leu Val Ala Thr Ala Trp His 1 5 10 15 Gly Gln Gly Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu Val Val             20 25 30 Lys Pro Gly Ala Thr Val Thr Leu Arg Cys Val Gly Asn Gly Ser Val         35 40 45 Glu Trp Asp Gly Pro Pro Ser Pro His Trp Thr Leu Tyr Ser Asp Gly     50 55 60 Ser Ser Ile Leu Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly 65 70 75 80 Thr Tyr Arg Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala                 85 90 95 Ile His Leu Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala             100 105 110 Gln Glu Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu         115 120 125 Leu Thr Asp Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg     130 135 140 Gly Arg Pro Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp His 145 150 155 160 Gly Phe Thr Ile His Arg Ala Lys Phe Ile Gln Ser Gln Asp Tyr Gln                 165 170 175 Cys Ser Ala Leu Met Gly Gly Arg Lys Val Met Ser Ile Ser Ile Arg             180 185 190 Leu Lys Val Gln Lys Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val         195 200 205 Pro Ala Glu Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys     210 215 220 Ser Ala Ser Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn 225 230 235 240 Asn Thr Lys Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg                 245 250 255 Tyr Gln Lys Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His             260 265 270 Ala Gly Asn Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys His Ser         275 280 285 Thr Ser Met Phe Phe Arg Val Val Glu Ser Ala Tyr Leu Asn Leu Ser     290 295 300 Ser Glu Gln Asn Leu Ile Gln Glu Val Thr Val Gly Glu Gly Leu Asn 305 310 315 320 Leu Lys Val Met Val Glu Ala Tyr Pro Gly Leu Gln Gly Phe Asn Trp                 325 330 335 Thr Tyr Leu Gly Pro Phe Ser Asp His Gln Pro Glu Pro Lys Leu Ala             340 345 350 Asn Ala Thr Thr Lys Asp Thr Tyr Arg His Thr Phe Thr Leu Ser Leu         355 360 365 Pro Arg Leu Lys Pro Ser Glu Ala Gly Arg Tyr Ser Phe Leu Ala Arg     370 375 380 Asn Pro Gly Gly Trp Arg Ala Leu Thr Phe Glu Leu Thr Leu Arg Tyr 385 390 395 400 Pro Pro Glu Val Ser Val Ile Trp Thr Phe Ile Asn Gly Ser Gly Thr                 405 410 415 Leu Leu Cys Ala Ala Ser Gly Tyr Pro Gln Pro Asn Val Thr Trp Leu             420 425 430 Gln Cys Ser Gly His Thr Asp Arg Cys Asp Glu Ala Gln Val Leu Gln         435 440 445 Val Trp Asp Asp Pro Tyr Pro Glu Val Leu Ser Gln Glu Pro Phe His     450 455 460 Lys Val Thr Val Gln Ser Leu Leu Thr Val Glu Thr Leu Glu His Asn 465 470 475 480 Gln Thr Tyr Glu Cys Arg Ala His Asn Ser Val Gly Ser Gly Ser Trp                 485 490 495 Ala Phe Ile Pro Ile Ser Ala Gly Ala His Thr His Pro Pro Asp Glu             500 505 510 Phe Leu Phe Thr Pro Val Val Ala Cys Met Ser Ile Met Ala Leu         515 520 525 Leu Leu Leu Leu Leu Leu Leu Leu Leu Tyr Lys Tyr Lys Gln Lys Pro     530 535 540 Lys Tyr Gln Val Arg Trp Lys Ile Ile Glu Ser Tyr Glu Gly Asn Ser 545 550 555 560 Tyr Thr Phe Ile Asp Pro Thr Gln Leu Pro Tyr Asn Glu Lys Trp Glu                 565 570 575 Phe Pro Arg Asn Asn Leu Gln Phe Gly Lys Thr Leu Gly Ala Gly Ala             580 585 590 Phe Gly Lys Val Val Glu Ala Thr Ala Phe Gly Leu Gly Lys Glu Asp         595 600 605 Ala Val Leu Lys Val Ala Val Lys Met Leu Lys Ser Thr Ala His Ala     610 615 620 Asp Glu Lys Glu Ala Leu Met Ser Glu Leu Lys Ile Met Ser His Leu 625 630 635 640 Gly Gln His Glu Asn Ile Val Asn Leu Leu Gly Ala Cys Thr His Gly                 645 650 655 Gly Pro Val Leu Val Ile Thr Glu Tyr Cys Cys Tyr Gly Asp Leu Leu             660 665 670 Asn Phe Leu Arg Arg Lys Ala Glu Ala Met Leu Gly Pro Ser Leu Ser         675 680 685 Pro Gly Gln Asp Pro Glu Gly Gly Val Asp Tyr Lys Asn Ile His Leu     690 695 700 Glu Lys Lys Tyr Val Arg Arg Asp Ser Gly Phe Ser Ser Gln Gly Val 705 710 715 720 Asp Thr Tyr Val Glu Met Arg Pro Val Ser Thr Ser Ser Asn Asp Ser                 725 730 735 Phe Ser Glu Gln Asp Leu Asp Lys Glu Asp Gly Arg Pro Leu Glu Leu             740 745 750 Arg Asp Leu Leu His Phe Ser Ser Gln Val Ala Gln Gly Met Ala Phe         755 760 765 Leu Ala Ser Lys Asn Cys Ile His Arg Asp Val Ala Ala Arg Asn Val     770 775 780 Leu Leu Thr Asn Gly His Val Ala Lys Ile Gly Asp Phe Gly Leu Ala 785 790 795 800 Arg Asp Ile Met Asn Asp Ser Asn Tyr Ile Val Lys Gly Asn Ala Arg                 805 810 815 Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Cys Val Tyr             820 825 830 Thr Val Gln Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile         835 840 845 Phe Ser Leu Gly Leu Asn Pro Tyr Pro Gly Ile Leu Val Asn Ser Lys     850 855 860 Phe Tyr Lys Leu Val Lys Asp Gly Tyr Gln Met Ala Gln Pro Ala Phe 865 870 875 880 Ala Pro Lys Asn Ile Tyr Ser Ile Met Gln Ala Cys Trp Ala Leu Glu                 885 890 895 Pro Thr His Arg Pro Thr Phe Gln Gln Ile Cys Ser Phe Leu Gln Glu             900 905 910 Gln Ala Gln Glu Asp Arg Arg Glu Arg Asp Tyr Thr Asn Leu Pro Ser         915 920 925 Ser Ser Arg Ser Gly Gly Ser Gly Ser Ser Ser Ser Glu Leu Glu Glu     930 935 940 Glu Ser Ser Glu His Leu Thr Cys Cys Glu Gln Gly Asp Ile Ala 945 950 955 960 Gln Pro Leu Leu Gln Pro Asn Asn Tyr Gln Phe Cys                 965 970 <210> 17 <211> 554 <212> PRT <213> Homo sapiens <400> 17 Met Thr Ala Pro Gly Ala Ala Gly Arg Cys Pro Pro Thr Thr Trp Leu 1 5 10 15 Gly Ser Leu Leu Leu Leu Val Cys Leu Leu Ala Ser Arg Ser Ile Thr             20 25 30 Glu Glu Val Ser Glu Tyr Cys Ser His Met Ile Gly Ser Gly His Leu         35 40 45 Gln Ser Leu Gln Arg Leu Ile Asp Ser Gln Met Glu Thr Ser Cys Gln     50 55 60 Ile Thr Phe Glu Phe Val Asp Gln Glu Gln Leu Lys Asp Pro Val Cys 65 70 75 80 Tyr Leu Lys Lys Ala Phe Leu Leu Val Gln Asp Ile Met Glu Asp Thr                 85 90 95 Met Arg Phe Arg Asp Asn Thr Pro Asn Ale Ile Val Gln Leu             100 105 110 Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr Glu         115 120 125 Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln     130 135 140 Leu Leu Glu Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu 145 150 155 160 Asp Lys Asp Trp Asn Ile Phe Ser Lys Asn Cys Asn Asn Ser Phe Ala                 165 170 175 Glu Cys Ser Ser Gln Asp Val Val Thr Lys Pro Asp Cys Asn Cys Leu             180 185 190 Tyr Pro Lys Ala Ile Pro Ser Ser Asp Pro Ala Ser Val Ser Pro His         195 200 205 Gln Pro Leu Ala Pro Ser Ala Pro Val Ala Gly Leu Thr Trp Glu     210 215 220 Asp Ser Glu Gly Thr Glu Gly Ser Ser Leu Leu Pro Gly Glu Gln Pro 225 230 235 240 Leu His Thr Val Asp Pro Gly Ser Ala Lys Gln Arg Pro Pro Arg Ser                 245 250 255 Thr Cys Gln Ser Phe Glu Pro Pro Glu Thr Pro Val Val Lys Asp Ser             260 265 270 Thr Ile Gly Gly Ser Pro Gln Pro Arg Pro Ser Val Gly Ala Phe Asn         275 280 285 Pro Gly Met Glu Asp Ile Leu Asp Ser Ala Met Gly Thr Asn Trp Val     290 295 300 Pro Glu Glu Ala Ser Gly Glu Ala Ser Glu Ile Pro Val Glu Gly 305 310 315 320 Thr Glu Leu Ser Pro Ser Arg Pro Gly Gly Gly Ser Met Gln Thr Glu                 325 330 335 Pro Ala Arg Pro Ser Asn Phe Leu Ser Ala Ser Ser Pro Leu Pro Ala             340 345 350 Ser Ala Lys Gly Gln Gln Pro Ala Asp Val Thr Gly Thr Ala Leu Pro         355 360 365 Arg Val Gly Pro Val Arg Pro Thr Gly Gln Asp Trp Asn His Thr Pro     370 375 380 Gln Lys Thr Asp His Pro Ser Ala Leu Leu Arg Asp Pro Pro Glu Pro 385 390 395 400 Gly Ser Pro Arg Ile Ser Ser Leu Arg Pro Gln Gly Leu Ser Asn Pro                 405 410 415 Ser Thr Leu Ser Ala Gln Pro Gln Leu Ser Ser Ser Ser Ser Ser Gly             420 425 430 Ser Val Leu Pro Leu Gly Glu Leu Glu Gly Arg Arg Ser Thr Arg Asp         435 440 445 Arg Arg Ser Pro Ala Glu Pro Glu Gly Gly Pro Ala Ser Glu Gly Ala     450 455 460 Ala Arg Pro Leu Pro Arg Phe Asn Ser Val Pro Leu Thr Asp Thr Gly 465 470 475 480 His Glu Arg Gln Ser Glu Gly Ser Phe Ser Pro Gln Leu Gln Glu Ser                 485 490 495 Val Phe His Leu Le Val Val Ser Val I Leu Val Leu Leu Ala Val             500 505 510 Gly Gly Leu Leu Phe Tyr Arg Trp Arg Arg Arg Ser His Gln Glu Pro         515 520 525 Gln Arg Ala Asp Ser Pro Leu Glu Gln Pro Glu Gly Ser Pro Leu Thr     530 535 540 Gln Asp Asp Arg Gln Val Glu Leu Pro Val 545 550 <210> 18 <211> 242 <212> PRT <213> Homo sapiens <400> 18 Met Pro Arg Gly Phe Thr Trp Leu Arg Tyr Leu Gly Ile Phe Leu Gly 1 5 10 15 Val Ala Leu Gly Asn Glu Pro Leu Glu Met Trp Pro Leu Thr Gln Asn             20 25 30 Glu Glu Cys Thr Val Thr Gly Phe Leu Arg Asp Lys Leu Gln Tyr Arg         35 40 45 Ser Arg Leu Gln Tyr Met Lys His Tyr Phe Pro Ile Asn Tyr Lys Ile     50 55 60 Ser Val Pro Tyr Glu Gly Val Phe Arg Ile Ala Asn Val Thr Arg Leu 65 70 75 80 Gln Arg Ala Gln Val Ser Glu Arg Glu Leu Arg Tyr Leu Trp Val Leu                 85 90 95 Val Ser Leu Ser Ala Thr Glu Ser Val Gln Asp Val Leu Leu Glu Gly             100 105 110 His Pro Ser Trp Lys Tyr Leu Gln Glu Val Glu Thr Leu Leu Leu Asn         115 120 125 Val Gln Gln Gly Leu Thr Asp Val Glu Val Ser Pro Lys Val Glu Ser     130 135 140 Val Leu Ser Leu Leu Asn Ala Pro Gly Pro Asn Leu Lys Leu Val Arg 145 150 155 160 Pro Lys Ala Leu Leu Asp Asn Cys Phe Arg Val Met Glu Leu Leu Tyr                 165 170 175 Cys Ser Cys Cys Lys Gln Ser Ser Val Leu Asn Trp Gln Asp Cys Glu             180 185 190 Val Pro Ser Pro Gln Ser Cys Ser Pro Glu Pro Ser Leu Gln Tyr Ala         195 200 205 Ala Thr Gln Leu Tyr Pro Pro Pro Pro Trp Ser Pro Ser Ser Pro Pro     210 215 220 His Ser Thr Gly Ser Val Arg Pro Val Arg Ala Gln Gly Glu Gly Leu 225 230 235 240 Leu Pro         

Claims (24)

CDRH1 comprising the sequence SYGMH (SEQ ID NO: 1), CDRH2 comprising the sequence VIWYDGSNKYYADSVKG (SEQ ID NO: 2), CDRH3 comprising the sequence GDYEVDYGMDV (SEQ ID NO: 3), CDRL1 comprising the sequence RASQGISNALA (SEQ ID NO: (SEQ ID NO. 15) comprising CDRL2 comprising the sequence QQFNSYPWT (SEQ ID NO: 6), and CDRL3 comprising the sequence QQFNSYPWT (SEQ ID NO: 6). 2. The method of claim 1, wherein the amino acid sequence:
VL containing AIQLTQSPSSLSASVGDRVTITCRASQGISNALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPWTFGQGTKVEIK (SEQ ID NO: 8)
And amino acid sequence:
QHQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGEGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYEVDYGMDVWGQGTTVTVAS (SEQ ID NO: 7)
&Lt; / RTI &gt; or a fragment thereof. The antibody or fragment thereof according to claim 1 or 2, which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10. 3. The antibody or a fragment thereof according to any one of claims 1 to 3, comprising two heavy chains each comprising the amino acid sequence of SEQ ID NO: 9 and two light chains each comprising the amino acid sequence of SEQ ID NO: 10. delete A pharmaceutical composition for the treatment of cancer, comprising the antibody of any one of claims 1 or 2 or a fragment thereof. The pharmaceutical composition according to claim 6, wherein the cancer is leukemia, breast cancer, endometrial cancer, prostate cancer, ovarian cancer, colorectal cancer, hepatocellular carcinoma, multiple myeloma, multiple myeloma or Hodgkin's lymphoma. The pharmaceutical composition according to claim 7, wherein the cancer is leukemia, breast cancer, endometrial cancer or prostate cancer. The pharmaceutical composition according to claim 6, wherein the antibody or fragment thereof is administered before, during, at the same time or after the start of a therapy using another anti-cancer therapeutic. 10. The method of claim 9, wherein the anti-cancer therapeutic agent is docetaxel, paclitaxel, Herceptin (Herceptin) ^® and the pharmaceutical composition is selected from the group consisting of doxorubicin. Comprising determining in vitro or in vitro the levels of CSF-1 or IL-34, or both, in a sample of a subject having cancer, wherein the sample comprises blood, serum, plasma, tumor cells and circulating tumor cells Lt; / RTI &gt;
Wherein an increase in the level of CSF-1 or IL-34, or both, relative to the level of CSF-1 or IL-34 in the non-cancerous individual, or both, is indicative that the subject is anti-CSF- Indicating that the candidate is a candidate for cancer therapy,
Wherein said antibody is an antibody according to claim 1 or 2, wherein said cancer is a candidate for anti-CSF-1R antibody-based cancer therapy. delete delete delete delete delete delete delete delete delete delete delete delete delete